JP2001505425A - Humanized anti-CD11a antibody - Google Patents

Abstract

  (57) [Summary] Disclosed are humanized anti-CD11a antibodies and various uses thereof. The humanized anti-CD1la antibody is specifically bound to the human CD11aI domain and has an IC50 (nM) value of up to about 1 nM to prevent adhesion of Jurkat cells to normal human epidermal keratinocyte-expressed ICAM-1; and And / or have an IC50 (nM) value of up to about 1 nM in a mixed lymphocyte response assay.

Description

DETAILED DESCRIPTION OF THE INVENTION                          Humanized anti-CD11a antibody                                Background of the Invention Field of the invention   The present invention relates generally to humanized anti-CD11a antibodies. Description of related technology   Lymphocyte function-related antigen 1 (LFA-1; CD11a / CD18) Answer: Included in leukocyte adhesion during cell interactions essential for inflammation (Larson Et al., Immunol. Rev. 114: 181-217 (1990)). LFA-1 is a β2 integrinpha Millie members and unique α subunits, CD11a and β subunits Knit, CD18, other common β2 integrin receptors Mac-1 and p1 Consists of 50,95. LFA-1 ligands include leukocytes, endothelial and dermal fibroblasts Intercellular adhesion molecule-1, ICAM-1, expressed above (Dustin et al., J. Immunol. 137: 245-254 (1986)), ICAM-2 expressed on dormant endothelium and lymphocytes (de Fougerol les et al. Exp. Med. 174: 253-267 (1991)), and expressed on monocytes and resting lymphocytes ICAM-3 (de Fougerolles et al., J. Exp. Med. 179: 619-629 (1994)).   Monoclonal antibodies (MAbs) against LFA-1 and ICAMs are , T cell activation (Kuypers et al., Res. Immunol. 140: 461 (1989)), T cell dependent B cells. Vesicle proliferation (Fischer et al., J. Immunol. 136: 3198-3203 (1986)), target cell lysis (Krensky J. et al. Immunol. 131: 611-616 (1983)), and adhesion of T cells to vascular endothelium (Lo et al., J. . Immunol. 143: 3325-3329 (1989)). Is shown. In mice, anti-CD11a MAbs are Resistance to cytoplasmic antigens (Tanaka et al., Eur. J. Immunol. 25: 1555-1558 (1995)), and Heart (Cavazzana-calvo et al., Transplantation 59: 1576-1582 (1995); Nakakura et al., T ransplantation 55: 1112-417 (1993)), bone marrow (Cavazzana-Calvo et al., Transplantati on 59: 1576-1582 (1995); van Dijken et al., Transplantation 49: 882-886 (1990)), Cornea (He et al., Invest. Opthamol. Vis. Sci. 35: 3218-3225 (1994)), Kojima (Nishiha La et al., Transp lantation Proc. 27: 372 (1995)) and thyroid (Talento et al., Transplantation 55:41). 8-422 (1993)) Induces prolonged survival of allografts.   In humans, anti-CD11a MAbs prevent graft failure after bone marrow transplantation (Fischer et al., Blood 77: 249-256 (1991); Stoppa et al., Transplant Intl. 4: 3-7 (19). 91)) and, in addition to corticosteroids and azathioprine, anti-CD11a MA Preliminary clinical studies of renal allografts treated prophylactically with b look promising (Hourmant et al., Transplantation 58: 377-380 (1994)). Current cure for transplant rejection Treatment with OKT3, murine anti-human CD3 MAb, and cyclosporin A Including OKT3 treatment is effective but has some undesirable side effects Its use is a cause of tumor necrosis resulting in fever, chills and gastrointestinal distress Child-α, interferon-γ, interleukin-2, and interleukin Resulting in the release of a number of cytokines, including -6 (for review, Parlevliet Et al., Transplant Intl. 5: 234-246 (1992); Dantal et al., Curr. Opin. Immunol. 3:74 0-747 (1991)). Cyclosporin A is effective but also has serious side effects (See Barry, Drugs, 44: 554-566 (1992) for reconsideration).                                Summary of the Invention   The present invention provides a humanized anti-CD11a antibody. A preferred antibody is human CD1 1a in the I-domain (eg, "Epitope MHM24 as defined herein" ") And / or about 1 x 10^-8With M or stronger affinity It binds to CD11a. In a preferred embodiment, the antibody is ICAM- Prevents Jurkat Cell Adhesion to Normal Human Epidermal Keratinocytes Expressing Escherichia coli Have an IC50 (nM) value of up to about 1 nM. Preferred humanized antibodies are mixed With an IC50 (nM) value of up to about 1 nM in the lymphocyte reaction (MLR) assay Those who do. This IC50 of the humanized antibody in the MLR assay is Significantly better than that of murine MAb 25.3, previously tested in vivo (Fischer et al., Blood 77: 249-256 (1991); Stoppa et al., Transplant Intl. 4: 3-7 (1991). Hourmant et al., Transplantation 58: 377-380 (1994)).   The humanized anti-CD11a antibody was obtained by converting the humanized antibody MHM24F (ab) -8 in FIG. D R1 (GYSFTGHWHN; SEQ ID NO: 10) and / or CDR2 (MIHPSDSETRYNQKFKD; sequence No. 11) and / or the amino acid sequence of CDR3 (GIYFYGTTYFDY; SEQ ID NO. 12) And / or the humanized antibody MHM24F (ab) -8 in FIG. CDR1 (RASKTISKYLA; SEQ ID NO: 13) and / or CDR2 (SGSTLQS; SEQ ID NO: : 14) and / or the amino acid sequence of CDR3 (QQHNEYPLT; SEQ ID NO: 15). Light chain variable region. In another embodiment, the antibody is humanized MHM2 4 comprising one or more amino acid sequence variants of CDRs of antibody F (ab) -8, A variant may have one or more amino acid insertions within and / or adjacent CDR residues and / or Deletions within or adjacent to CDR residues and / or substitutions of CDR residues (such variants With substitutions as a suitable type of amino acid replacement to produce So Is about 1 × 10^-8The binding affinity of human CD11a up to M Will have.   In a preferred embodiment, the humanized antibody has the amino acid sequence of SEQ ID NO: 2. 1 and / or the sequence number of the humanized antibody MHM24F (ab) -8 in FIG. No .: Heavy chain variable region comprising the amino acid sequence of 5 and / or amino acid sequence variation thereof Includes the body.   As described herein, rhesus monkey CD11a (ie, a "rhesus" antibody) Binds the human CD11a antigen to confer the ability to bind, but Humanized antibodies not significant with rhesus monkey CD11a antigen can be engineered later It is said to be capable. In this embodiment, an antibody that binds rhesus monkey CD11a May comprise, for example, the CDR2 amino acid sequence in SEQ ID NO: 23. Other CDRs Can be the same as those for the humanized MHM24 antibody F (ab) -8. Hide The antibody optionally linked to a light chain comprising the amino acid sequence of SEQ ID NO: 2; The amino acid sequence of the "rhesus" heavy chain in SEQ ID NO: 24 may be provided. The antibody Various forms are contemplated herein. For example, the anti-CD11a antibody is a full-length antibody ( For example, having a human immunoglobulin constant region) or an antibody fragment (e.g., F (ab ')_Two). Further, the antibody is labeled with a detectable label and It can be immobilized and / or conjugated to a foreign compound (such as a cytotoxic agent).   Diagnostic and therapeutic uses for the antibodies are contemplated. In one diagnostic application , The present invention relates to a method for preparing a sample suspected of containing a CD11a protein in an anti-CD11a antibody. CD1 comprising exposing the sample and measuring the binding of the antibody to the sample Methods are provided for measuring the presence of the 1a protein. For this use, Ming comprises an antibody for using the antibody for detecting the CD11a protein Provide kit and instructions.   The invention further includes: an isolated nucleic acid encoding the antibody; a vector comprising the nucleic acid. Controls the sequence recognized by the host cell transformed with the vector. Operably linked to a host cell comprising the vector; the nucleic acid Culturing the host cell so that is expressed, and optionally, Producing the antibody comprising recovering the antibody (e.g., from a host cell culture medium). Provide a method for The present invention also relates to pharmaceutically acceptable humanized anti-CD11a antibodies. Also provided is a composition comprising a carrier or excipient. This for therapeutic use Is sterile and can be lyophilized. The invention further relates to a mammal comprising a human LFA-1 mediated disease, comprising administering a pharmaceutically effective amount of a humanized CD11a antibody Methods for treating a mammal afflicted with A. For such therapeutic uses Other immunosuppressive agents or adhesion molecule antagonists (eg, another LFA-1 Agonist or VLA-4 antagonist) before the humanized anti-CD11a antibody. May be co-administered to the mammal, either later, or simultaneously.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1A shows the murine MHM24 light chain (SEQ ID NO: 1), humanized MHM24 F (ab) -8 light chain (SEQ ID NO: 2), human consensus arrangement of subgroup light chain κI (humκI) The amino acid sequence of the row (SEQ ID NO: 3) is shown.   FIG. 1B shows murine MHM24 heavy chain (SEQ ID NO: 4), humanized MHM24 F (ab) -8 heavy chain (SEQ ID NO: 5), human consensus arrangement of heavy chain subgroup III (humIII) Sequence (SEQ ID NO: 6) and the “rhesus monkey” antibody variant weight chain of the example (SEQ ID NO: 2) The amino acid sequence of 4) was shown.   1A and 1B, a hypervariable region based on sequence hypervariability (Kabat et al., Sequ ences of Proteins of Immunological Interest. 5th Ed. Public Health Servi ce, National Institutes of Health, Bethesda, MD. (1991)) And a hypervariable loop based on the structure of the F (ab) -antigen complex (Chothia et al., (Nature 342: 8767 (1989)) is italicized. Residue numbering is a, b, and c According to Kabat et al.   FIG. 2 shows human CD11a I-domain (SEQ ID NO: 7) and rhesus monkey CD11. a The sequence of the I-domain (SEQ ID NO: 8) is shown. β chain and α helix are underlined And Qu et al., Proc. Natl. Acad. Sci. 92: 10277-10281 (1995) Is done. The rhesus monkey I-domain sequence (rhCD11a) And only four differences. MHM24 MAb (SEQ ID NO: 9) Binding epitopes are shown in boldface type (Champe et al., J. Biol. Chem. 270: 1388-1). 394 (1995)).   FIG. 3 shows murine MHM24 (closed circle), chimeric MHM25 (open triangle), human MHM24 (HuIgG1) (filled square) and human IgG1 isotype control Roll (+) suppresses human Jurkat T cells to normal human keratinocytes Represents a system. Percent binding was measured by fluorescence of labeled Jurkat cells.   Figures 4A-4C show rhesus monkey lymphocyte response to normal human keratinocytes. (FIG. 4A) Rhesus monkey against recombinant human ICAM-1 coated on plate Rhesus monkeys / humans on lymphocytes (FIG. 4B) and normal human keratinocytes Inhibition of binding of the transfected 293 cells (FIG. 4C) was demonstrated. Rhesus Monkey-bound MHM24 (RhIgG1) (packed squares), anti-CD18MHM23 (packed squares) Circles), human IgG1 isotype control (+) (FIGS. 4A and 4C), and murine Suppression by IgG1 isotype control (+) (FIG. 4B). The percent join is Measured by fluorescence of labeled lymphocytes (FIGS. 4A and B) or labeled 293 cells (FIG. 4C) did.   FIG. 5 shows that the human lymphocyte reaction assay (MLR) was performed in murine MHM24 (clogged). Circles), humanized MHM24 (HuIgG1) (filled squares), and humanized isotype Ig Shown to be blocked by G control (full diamond) . Percent stimulation index (% SI) is given for maximal response in the absence of MAb. The ratio of the response at the given MAb concentration. The data contains at least two different stimuli 9 is an illustration of a multiple assay using a / response pair.                        Detailed Description of the Preferred Embodiment I. Definition   Unless otherwise indicated, as used herein, the term “CD11a” refers to any term For the α subunit of LFA-1 from mammals, but preferably from humans I do. The CD11a can be separated from molecules of natural source or can be synthesized (Eg, using recombinant DNA technology). For human CD11a The amino acid sequence is described, for example, in EP 362 526B1.   The term "I-domain" is described in Champe et al. Biol. Chem. 270: 1388-1394 (1995) and And / or Qu et al., Proc. Natl. Acad. Sci. 92: 10277-10281 (1995). Regarding that area of the child. Human CD11a I-domain (SEQ ID NO: 7) and rhesus The amino acid sequence of the monkey CD11a I-domain (SEQ ID NO: 8) is shown in FIG. Illustrated in FIG.   As used herein, the term “epitope MHM24”, unless otherwise indicated, refers to A region in the I-domain of human CD11a that binds to the HM24 antibody (see below) Related. This epitope comprises the amino acid sequence of SEQ ID NO: 9, and optionally, the CD 11a and / or other amino acid residues of CD18.   The term "LFA-1 mediated disease" involved the LFA-1 receptor on lymphocytes. It relates to pathological conditions caused by cell adhesion interactions. Such a disease Examples of T cell inflammatory responses, such as inflammatory skin diseases including psoriasis; Responses associated with disease (such as Crohn's disease and ulcerative colitis); adult respiratory distress symptoms Group; dermatitis; meningitis; encephalitis; uveitis; allergic conditions such as eczema and asthma Conditions involving the infiltration of T cells and chronic inflammatory responses; Inflammation (including poison ivy and poison oak); atherosclerosis; leukocyte adhesion failure; Rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes, multiple sclerosis, Nard syndrome, autoimmune thyroiditis, experimental autoimmune encephalomyelitis, Sjogren Syndrome, juvenile onset diabetes, and cytokines and T typically found in the vessels Immune responses associated with lymphocyte-mediated delayed hypersensitivity, sarcoidosis, Autoimmune diseases such as spontaneous myositis, granulomatosis and vasculitis; pernicious anemia; chronic obstructive lung Diseases (COPD); bronchitis; insulinitis; rhinitis; hives; glomerulonephritis; leukocytes Leak CNS inflammatory disease; multi-organ secondary injury to sepsis or trauma Climate group; autoimmune hemolytic anemia; myethemia gravis; Complex-mediated diseases; nephrotic syndrome; malignant diseases (eg, chronic lymphocytic white B-cell malignancies such as hematologic or hairy cell leukemia); graft-versus-host or host All forms of transplant disease, including anti-grafts; HIV and rhinovirus infection; lung lines Including the conversion of tumor cells into secondary organs of fibrosis.   The term "immunosuppressant," as used herein for accompanying treatment, refers to the Anything that acts to suppress or mask the host's immune system while the piece is being transplanted About quality. This downregulates autoantigen expression, suppressing cytokine production. Or substances that modulate or suppress or mask MHC antigens. Would. An example of such an agent is 2-amino-6-aryl-5-substituted pyrimidine (US No. 4,665,077), azathioprine (or cyclophosphamide, if Bromocriptine; glutarua) Rudehydride (masks MHC antigens as described in U.S. Pat. No. 4,120,649) Anti-idiotypic antibodies for MHC antigens and fragments; cyclos Porin A; glucocorticoids such as prednisone, methylprednisone, and And steroids such as dexamethasone; anti-interferon-γ, -β, or -Cytokine or cytokine receptor antagonist containing -α antibody; antitumor Tumor necrosis factor α antibody; anti-tumor necrosis factor β antibody: anti-interleukin-2 antibody and anti-tumor necrosis factor-2 antibody IL-2 receptor antibody; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; Body, preferably an anti-CD3 or anti-CD4 / CD4a antibody; including an LFA-3 binding region. Peptide (WO 90/08187 published July 26, 1990); Streptokinase; T GF-β; streptodornase; RNA or DNA from a host; FK506; RS-61443; deoxyspagarin; rapamycin; T cell receptor (US No. 5,114,721); T cell receptor fragment (Offner et al., Science 251). : 430-432 (1991); WO 90/11294; and WO 91/01133); and T-cells such as T10B9. Vesicle receptor antibody (EP340,109). These agents are used simultaneously with the CD11a antibody. Or administered at discrete times, and is the same or as described in the art. Used at lower dosages. Preferred ancillary immunosuppressants are based on patient history Also implemented Based on a number of factors, including the form of the disease being treated, including the form of transplant Yes, but generally generally preferred the agent is cyclosporin A, glucosporin Corticoids (groups are also preferably prednisone or methylprednisone), OKT- 3 monoclonal antibody, azathioprine, bromocriptine, heterologous anti-lymphocyte An agent selected from roblin or a mixture thereof.   "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. . Those in need of treatment are the same as those in whom the disease is to be prevented , Including those who already have the disease.   A “mammal” for the purpose of treatment is a domestic animal such as a human, dog, horse, cat, cow, or the like. Breeding or domestic animals, and mammals, including zoos, sports, or pets For any animal that is classified. Preferably, the animal is a human.   As used herein, the term "graft" refers to a donor for transplantation within a subject. It relates to the obtained biomaterial. Grafts include islet cells and nerve-derived cells (e.g., Schwann Cells), such as neonatal amniotic membrane, bone marrow, hematopoietic progenitor cells Tissue, skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, vascular organs (e.g. (Eg, blood vessels or esophagus). Vascular organs, esophagus, blood It can be used in place of a damaged part of a tube or a tube. Skin graft In addition, it blocks certain injuries in the damaged intestine, such as-or diaphragmatic hernia It can be used in particular for preparation. The graft can be from a cadaver or a living donor. Obtained from any mammalian source, including humans. Preferably, the The graft is an organ such as bone marrow or heart, and the donor and host of the graft are , HLA class II antigen.   The term "donor" as used herein refers to a dead or live mammal from which a graft is obtained. Regarding animal species. Preferably, the donor is a human. Human donor with major blood Because group crossing is a potential barrier to allogeneic transplant survival, -Related in volunteers who are characteristic of the same test and are the same major ABO blood group Donors are preferred. However, it does not allow, for example, into A, B or AB recipients. Transplantation of a type O donor kidney is possible.   The term “transplant” and variations thereof are used when the transplant is syngeneic (where the donor is When Recipients are generally identical), allogeneic (where the donor and recipient are of genetic origin Different but the same species) or heterogeneous (where the donor and recipient are from different species) The insertion of a graft into a host. Thus, typical In a typical scenario, the host is human and the graft is the same or different genetic origin. Syngeneic grafts obtained from the source human. In another scenario, the implant is Such as the heart of a baboon transplanted into a recipient host, and, for example, a porcine heart valve, or Phylogenetic spreads such as animal beta islet cells or neurons transplanted into a human host Obtained from a different species than the one to which it is transplanted, including animals from well-separated species .   "Increasing the resistance of the transplanted graft" by the host means that Prolong the survival of the piece, ie it will well tolerate transplantation from others And suppressing the host immune system.   "Intermittent" or "periodic" dosing can be for a period of time, and for one or more Separation or continuous administration at regular intervals, which is preferred.   The "selected resistance" of a disease is the host's resistance to specific factors that cause the disease. Tolerated by the immune system, but rejecting a second allogeneic or xenograft To maintain the capacity of the host. Preferably, the resistance is such that the immune system It's like leaving a complete.   The term "LFA-1 antagonist" refers to the interaction of LFA-1 with ICAM-1. Molecules that act as competitive inhibitors for Examples of such molecules are , CD11a (eg, humanized anti-CD11a described herein) or CD18 or both Alternatively, antibodies to ICAM-1 and other molecules such as peptides (eg, pseudo-peptides) Antibodies directed against any of them.   The term "VLA-4" is a competitive inhibitor of VLA-4 interaction with VCAM. Related to molecules that act as a ligand. Examples of such molecules are VLA-4 or VLA-4 Directed against either CAM and other molecules (eg, pseudopeptide antagonists) Antibodies.   The term `` antibody '' refers to monoclonal antibodies (including full-length monoclonal antibodies), Riclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and preferably The broadest understanding and specific inclusion of antibody fragments as long as they exhibit biological activity Used in doing.   An "antibody fragment" is a portion of a full-length antibody, generally It has a variable region for it. Illustrative examples of antibody fragments include Fab, Fab ', F (a b ')<sub>Two</sub>And Fv fragments; dimers; linear antibodies; single-chain antibody molecules; Includes multispecific antibodies formed from fragments.   The term "monoclonal antibody" as used herein refers to a substantially homogeneous antibody. For antibodies obtained from a population, i.e., individual antibodies contained in the Except for the possibility of spontaneous mutations that may be present. monoclonal Antibodies are highly specific, being directed against a single antigenic site. In addition, they typically include different antibodies directed against different determinants (epitope). Each monoclonal antibody against a normal (polyclonal) antibody preparation. Are directed against a single determinant on the antigen. The modifier "monoclonal" Exhibit characteristics of the antibody as obtained from a substantially homogeneous population of antibodies, and For composing requesting production of antibodies by some special method Absent. Monoclonal antibodies to be used in accordance with the present invention are described in Kohler et al., Nature 2 56: 495 (1975). Or can be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). See). The "monoclonal antibody" can also be described, for example, in Clackson et al., Nature 352: 624-. 628 (1991) and Marks et al. Hol. Biol. 222: 581-597 (1991) Alternatively, it can be isolated from a phage antibody library.   A monoclonal antibody here is a unique species or unique antibody class or subclass. Homology identical or identical to a sequence in an antibody obtained from a member of the family A "chimeric" antibody (immunoglobulin) in the heavy and / or light chain While specifically comprising, the rest of the chain is of another species or another antibody class or subclass. Homologues identical or identical to sequences in antibodies obtained from those belonging to As well as the fragmentation of such antibodies, as long as they exhibit the desired biological activity. (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. . USA 81: 6851-6855 (1984)).   As used herein, the term "hypervariable region" is responsive for antigen binding Regarding the amino acid residues of the antibody. The hypervariable region may be a “complementary detection region” or “CD R '' (ie, residues 24-34 (L1), 50-56 (L2 ) And 89-97 (L3) and 31-35 (H1), 50-65 (H2) and 95-1021 (H3) in the heavy chain variable region; K abat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / Or "hypervariable loop" (i.e., residues 26-32 (L1), 50-: 52 (L 2) and 91-96 (L3) and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable region; Ch othia and Lesk J. Mol. Biol. 196: 901-917 (1987)). “Framework” or “FR” residues are hypervariable region residues as described herein. Are those variable region residues other than.   A `` humanized '' form of a non-humanized (e.g., murine) antibody is derived from a non-human immunoglobulin. Chimeric antibody containing the minimum sequence. For the most part, humanized antibodies are Hypervariable region residues have the desired specificity, affinity and capacity in mice, rat Hypervariable region residues from a non-human species such as a human, rabbit or non-human primate. The human immunoglobulin (receptor antibody) is replaced (donor antibody). Some examples Wherein the Fv framework region (FR) residues of the human immunoglobulin are identical. Is replaced by a non-human residue. In addition, humanized antibodies are Or residues not found in the donor antibody. These fixes are anti- Performed to further refine the body's efficiency. Generally, the humanized antibody is a non-human All or substantially all of the hypervariable loop corresponding to that of an immunoglobulin; And all or substantially all of the FR regions are those of a human immunoglobulin sequence Would comprise at least one, and typically substantially all, of the variable regions U. The humanized antibody optionally may be an immunoglobulin, typically a human immunoglobulin assay. It will also include at least a portion of the normal region (Fc). For further details, see Jones et al. Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Pre. sta, Curr. Op. Struct. Biol. 2: 593-596 (1992).   “Single-chain Fv” or “sFv” antibody fragments are those that express the V_HAnd V_LDomain and And these domains are present in a single polypeptide chain. Generally, the F The v polypeptide is V_HAnd V_LDesirable structure for antigen binding between domains It further comprises a peptide linker that provides the sFv for formation. Reconsideration of sFv The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and M oore eds. See Pluckthun in Springer-Verlag, New York, pp. 269-315 (1994). .   The term "diabodies" is a small antibody with two antigen binding sites With respect to fragments, the fragments may comprise the light chain variable region (V_L Heavy chain variable region (V_H) And (V_H-V_L). Two domains on the same chain By using a very short linker that allows pairing between the regions, the region is Compelling it to pair with the complementary domain of another chain and To create Diabodies are described, for example, in EP 404,097; WO 93/11161; and Holling. er et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Will be posted.   As used throughout this application, the expression “linear antibody” is defined by Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995). Briefly, these Antibodies comprise a pair of tandem Fd segments (V_H-C_H1 -V_H-C_H1) is provided. Linear antibodies can be bispecific or multispecific.   An “isolated antibody” is identified and separated from components of its natural environment and And / or one that has been recovered. Contaminants in its natural environment are diagnostic for the antibody. Or materials that would interfere with therapeutic use, such as enzymes, hormones and other proteins. Or it may contain non-proteinaceous solutes. In a preferred embodiment, the antibody comprises: Greater than 95% by weight of the antibody as measured by the Lie method, most preferably To 99% by weight or more, (2) N- by using spinning cup sequenator To a degree sufficient to obtain at least 15 residues of the terminal or intermediate amino acid sequence. Or (3) Coomassie blue or, as appropriate, silver staining, under reducing or non-reducing conditions. Will be purified to homogenize by SDS-PAGE. Anti isolated Whether the body will be absent of at least one component of the antibody's natural environment And antibodies in situ within recombinant cells. Usually, however, isolated anti The body will be prepared by at least one purification step.   As used herein, the term “epitope tagging” translates to “epitope tag”. The combined anti-CD11a antibodies. Epitope tag polypeptides Antibodies that do not interfere with the activity of the CD11a antibody. It has enough residues to provide an epitope for something short. The epi Tope tags preferably have antibodies that substantially cross-react with other epitopes. Unique enough to not respond. Suitable tag polypeptides are generally Both have 6 amino acid residues, usually 8-50 amino acid residues (preferably 9-30 residues). Group). Illustrative examples are the flu HA tag polypeptide and its antibody 12CA5. (Hield et al., Mol. Cell. Biol. 8: 2159-2165 (1988)); c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (Evan et al., Hol. Ce. ll. Biol. 5 (12): 3610-3616 (1985)); and herpes simplex virus glycoployte In-D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3 (6): 547- 553 (1990)). In some embodiments, the epitope tag is "salvae". Direceptor binding epitope ".   As used herein, the term "salvage receptor binding epitope" refers to Ig G molecules (eg, IgG₁, IgG_Two, IgG_ThreeOr IgG_FourEpitope of Fc region To increase plasma half-life of IgG molecules in vivo Can respond to   The term “cytotoxic agent” as used herein refers to inhibiting or preventing the function of a cell. And / or substances that cause cell degradation. The term radioactive iso Tope (I¹³¹, I¹²⁵, Y⁹⁰And Re¹⁸⁶), Chemotherapeutic agents, and bacterial, moldy, Contains toxic substances, such as plant or animal enzymatically active poisons, or fragments thereof. Is intended.   "Chemotherapeutic agents" are compounds useful in the treatment of cancer. Examples of chemotherapeutic agents Means adriamycin, doxorubicin, 5-fluorouracil, cytosine Vinoside (`` Ara-C ''), cyclophosphamide, thiotepa, taxotere (docetaxe l), busulfan, cytoxin, taxol, methotrexate, cisplatin , Melphalan, vinblastine, bleomycin, etoposide, ifosuf Amid, mitomycin C, mitozantrone, vincristine, binoruel Bottle, carboplatin, teniposide, daunomycin, carminomycin, ami Nopterin, dactinomycin, mitomycin, esperamicin (U.S. Pat. , 675,1 No. 87), other related to melphalan and nitrogen mustard Including things.   The term “prodrug”, as used in the present invention, precedes the pharmaceutically active substance. With respect to the precursor or derivative form, i.e. less cytotoxic to tumor cells compared to the parent drug And can be enzymatically activated or converted to a more active form . For example, Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al. "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug  See Delivery, Borchardt et al. (Ed.), Pp. 247-267, Humana Press (1985). The present invention Prodrugs of, but not limited to, phosphate-containing prodrugs , Thiophosphate-containing prodrugs, sulfate-containing prodrugs, pepti -Containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs , Β-lactam-containing prodrug, optionally substituted phenoxyacetamide pro Drugs or optionally substituted prodrugs containing phenylacetamide Luorocytosine and other 5- that can be converted to more active cytotoxic free drugs Includes fluorouridine prodrugs. Prodora for use in the present invention Illustrative examples of cytotoxic drugs that can be derivatized to the egg type include, but are not limited to, those described above. Contains chemotherapeutic agents.   As used herein, the term “label” refers to a test directly or indirectly conjugated to an antibody. It relates to a compound or composition that can be delivered. The label is detectable by itself. (E.g., radioisotope labeling or fluorescent labeling) or in the case of enzyme labeling. And a catalytic chemical replacement of the detectable substrate compound or composition.   Non-aqueous matrix such that the alternation of the invention is adherent by "solid phase" Is meant. Examples of solid phases included here are glass (e.g., controlled pore glass). ), Polysaccharides (e.g., agarose), polyacrylamide, polystyrene Partially or completely formed of polyvinyl alcohol and silicone including. In certain embodiments, based on the context, the solid phase is an assay plate. Can be included; in others it is a purification column (eg, Affinity chromatography column). This term is also used in U.S. Pat. In 5,149 It also includes a discontinuous solid phase of separable particles, such as those described.   "Liposome" refers to a drug for mammals (the anti-CD11a described herein and any Various types of fats that are useful for the delivery of Small vesicles composed of carbohydrates, phospholipids and / or surfactants. The re The components of the posome are typically arranged in a bilayer morphology resembling the lipid arrangement of biofilms .   An "isolated" nucleic acid molecule is one to which it is normally bound in the natural source of the antibody nucleic acid. Nucleic acid identified and separated from at least one contaminating nucleic acid molecule by Is a molecule. An isolated nucleic acid molecule is one in which it is found in nature. Other than shape or setting. Isolated nucleic acid molecules are therefore Distinguishing from nucleic acid molecules such as those present in cells. However, the separated nucleus An acid molecule is an arrangement in which, for example, its nucleic acid molecule differs in chromosome from that of a natural cell. In the case of, it includes a nucleic acid molecule contained in a cell in which the antibody is normally expressed.   The expression "control sequence" refers to an operably linked code in a unique host organism. DNA sequences necessary for the expression of the functionalized sequence. Suitable for prokaryotes Control sequences include, for example, promoters, optional operator sequences, and Includes bosome binding site. Eukaryotes contain the promoter, polyadenylation signal It is known to utilize files and enhancers.   When a nucleic acid is placed into a functional relationship with another nucleic acid sequence, "Combined as possible." For example, presequence DNA or secretion leaders may be If it is expressed as a preprotein that participates in the secretion of the polypeptide Operably linked to DNA for a polypeptide; promoter and enhancer Operatively binds to the coding sequence if it affects the transcription of the sequence. The combined ribosome binding site is the site where it is placed to facilitate translation. Is operably linked to the coding sequence. Generally, "operable" DNA sequences that are contiguous and, in the case of a secretory leader, Contact and in the reading phase. However, Enha The sensors do not have to be adjacent. Joins are useful at restricted sites. Achieved by ligation. If no such site exists, the synthetic oligo Nucleotide adapters or linkers are used according to normal practices. You.   As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably. And all such definitions include the resultant. Scratch The terms "transformation" and "transformed cells" And cultures obtained therefrom. It also means that all the results Perfect identity in DNA content due to deliberate or accidental mutations It is understood that it is not necessary. Screen in uniquely transformed cells Mutations having the same function or biological activity. Individual definitions Where intended, it will be clear from the context. II. BEST MODE FOR CARRYING OUT THE INVENTION   A. Antibody preparation   Methods for humanizing non-human CD11a antibodies are described in the Examples below. . To humanize an anti-CD11a antibody, a non-human antibody starting material is prepared. So Exemplary methods for purifying antibodies such as are described in the sections below. .         (I) Preparation of antigen   The CD11a antigen to be used for the preparation of the antibody is, for example, CD11a or CD11a. A soluble form of the extracellular domain of the 11a fragment (eg, the CD11a I domain CD11a fragment with "MHM24 epitope" like fragment ). Alternatively, the cell expressing CD11a on its cell surface is an antibody Can be used to generate Such cells contain CD11a and optionally C It can be transformed to express D18, or other naturally occurring cells (eg, human Empablast cells, Hildreth et al., Eur. J. Immunol. 13: 202-208 (1983)) or ja -Cut cells (see Examples below). CDs useful for generating antibodies Other forms of 11a will be apparent to those skilled in the art.         (Ii) Polyclonal antibody   Polyclonal antibodies can be derived from multiple subcutaneous (sc) or related antigens and adjuvants. It is preferably raised by intraperitoneal (ip) injection. For example, keyhole rimpet hemo Cyanine, serum albumin, bovine thyroglobulin, or a bifunctional or inducing agent, eg For example, maleimidobenzoylsulfosuccinimide ester (via cysteine residue N-hydroxysuccinimide (via lysine residue), glutaraldehyde Hyd, succinic anhydride, SOCl_TwoOr R¹N = C = NR, where R and R¹Is different Should be immunized, such as soybean trypsin inhibitor using an alkyl group Can be useful for conjugating related antigens to proteins that are immunogenic in You.   The animal may, for example, receive the protein or protein together with 3 volumes of Freund's complete adjuvant. 100 μg or 5 μg of conjugate (for rabbit or mouse, respectively) Antigen, immunogenic conjugate, or binding by injecting the solution subcutaneously with the Immunization against the derivative due to One month later, the animals were skinned at multiple sites 1/5 to 1/10 of original amount in Freund's complete adjuvant by lower injection Of the peptide or conjugate. After 7 to 14 days, the animals are released Blood is collected and the serum is analyzed for antibody titers. Animals up to titer plateau Boosted. Preferably, the animals are boosted by conjugation of the same antigen but , To different proteins and / or through different crosslinkers. Joint body Alternatively, it can be made in recombinant cell culture as a protein fusion. Also, alum Such aggregating agents are suitably used to increase the immune response.         (Iii) Monoclonal antibody   Monoclonal antibodies were first described by Kohler et al., Nature, 256: 495 (1975). It can be made using the hybridoma method described, or can be made using recombinant DNA methods (U.S. Pat. No., 816,567).   In the hybridoma method, mice, hamsters or macaque monkeys Suitable host animals will specifically bind to the protein used for immunization. Above to elicit lymphocytes that produce or may produce antibodies Immunized as you did. Alternatively, the lymphocytes are immunized in vitro. Can be The lymphocytes then form polyethylene to form hybridoma cells. It is fused with myeloma cells using a suitable fusion agent such as glycol (Goding, Mon. oclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 19 86)).   The hybridoma cells thus prepared are seeded and unfused, parental bone marrow. Suitably containing one or more substances that inhibit the growth or survival of the tumor cells. Cultured in the appropriate medium. For example, if the parental myeloma cell is Lacks guanguanine phosphoribosyltransferase (HGPRT or HPRT) If so, the medium for a typical hybridoma is the growth of HGPRT-deficient cells. May contain substances that prevent length, hypoxanthine, aminopterin and thymidine (HAT medium).   Preferred myeloma cells are those that fuse the selected antibody-producing cells efficiently and are stable and high-reactive. Those that support bell production and are sensitive to media such as HAT media. You. Among these, the preferred myeloma cell line is the Salk Institute Cell Distributi on Center, MOP-21 available from San Diego, California, USA Those obtained from MC-11 mouse tumors and American Type Culture Collect SP-2 or X63-Ag available from ion, Rockville, Maryland, USA A murine myeloma line such as 8-653 cells. Human myeloma and mouse-human differences Seed myeloma cell lines have also been described for the production of human monoclonal antibodies (K ozbor, J. Immunol. , 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Produc. tion Techniques and Applications, pp.51-63 (Harcel Dekker, Inc., New York , 1987)).   The medium in which the hybridoma cells are growing is made up of a monoclonal medium directed against the antigen. And analyzed for the production of local antibodies. Preferably, the hybridoma cells The binding specificity of monoclonal antibodies produced by immunoprecipitation or radio In vivo assays such as immunoassays (RIAs) or enzyme-linked immunosorbent assays (ELISAs) It is measured by a binding assay in vitro.   The binding affinity of the monoclonal antibody can be determined, for example, by the method of Hunson et al., Anal. Biocllem., 107 : 220 (1980).   Hybridoma cells produce antibodies of desired specificity, affinity, and / or activity Clones, the clones are subcloned by limited dilution. And can be propagated by standard methods (Goding, Monoclonal Antibodies : Principles and Practice, pp.59-103 (Academic Press, 1986)). For this purpose Suitable culture media include, for example, D-MEM or RPMI-1640 medium. In addition hand, Hybridoma cells can be grown in vivo as ascites tumors in animals.   The monoclonal antibody secreted by the subclone is, for example, protein A -Sepharose, hydroxyapatite chromatography, gel electrophoresis, Analysis or normal immunoglobulin purification, such as affinity chromatography. Depending on the manufacturing method, it is appropriately separated from the culture medium, ascites fluid, or serum.   DNA-encoded monoclonal antibodies can be readily isolated and used in standard procedures (eg, For example, it specifically binds to the genes encoding the heavy and light chains of the monoclonal antibody. (By using oligonucleotide probes that can be The columns are determined. The hybridoma cells serve as a suitable source of such DNA. Serve. In a single separation, the DNA is isolated from the monoclonal host in a recombinant host cell. Escherichia coli, apes COS cells, Chinese ham to obtain antibody synthesis Do not produce star egg (CHO) cells or otherwise immunoglobulin proteins. Can be placed in an expression vector that is transferred into a host cell, such as a myeloma cell. . Recombinant production of antibodies will be described in more detail below.           (Iv) Humanized and amino acid sequence variants   The following example describes a procedure for humanization of an anti-CD11a antibody. A certain fruit In one embodiment, to produce amino acid sequence variants of the humanized antibody, Is desirable if it improves the binding affinity or other biological properties of the humanized antibody And   The amino acid sequence variant of the humanized anti-CD11a antibody is humanized anti-CD11a antibody D By introducing appropriate nucleotides that change NA or for peptide synthesis It is thus prepared. Such a mutant is for humanized anti-CD11a F (ab) -8 Deletion of residues in the amino acid sequence shown in (for example, like SEQ ID NOs: 2 and 5), And / or insertions and / or substitutions. Created to occur in the final structure Any combination of deletions, insertions, and substitutions that make the final structure desirable Provided to own. The modified amino acids also appear at the glycosylation site. Alter the post-translation process of the humanized anti-CD11a antibody, such as altering its number or position. I can get it. Humanized anti-CD11a antibody polypeptes in a suitable arrangement for mutagenesis Useful methods for identifying certain residues or regions of the tides are described in Cunningham and Wells Sc ience, 244: 1081-1085 (1989), "Alanine scanning mutagenesis. ". Here, a group of residues or target residues is identified (e.g., arg, asp, his , lys, and glu) and amino acids by the CD11a antigen. Neutral or negatively charged amino acids that affect the interaction (most preferred Is replaced by alanine or polyalanine). Functional choice for the replacement The amino acid configuration that indicates the sex may be at the site of further introduction or substitution or at that site. Purified by other variants. Thus, introducing an amino acid sequence variant Sites for mutations are predetermined, while the nature of the mutations themselves must be predetermined. No need. For example, to analyze the performance of a mutation at a given site, Humanized antisense or random mutagenesis has been introduced and expressed at the target codon or region CD11a antibody variants are screened for the desired activity.   Amino acid sequence inserts are similar to intersequence inserts of single or multiple amino acid residues. Within the length from 1 residue containing 100, or more residues to the polypeptide Amino- and / or carboxy-terminal fusions. An example of a terminal insert is N -Humanized anti-CD11a antibody with terminal methionyl residue or epitope tag The combined antibody. Other insertional variants of the humanized CD11a antibody molecule are those of the antibody. Enzyme or polypeptide humanized anti-CD11a antibody N that increases serum half-life Or a fusion to the C-terminus (see below).   Another type of variant is an amino acid substitution variant. These mutants are removed Having at least one amino acid residue in a humanized anti-CD11a antibody molecule Residue is inserted in its place. The most important sites for alternative mutagenesis are Includes a variable loop, but FR alternations are also contemplated. Table I in Examples described below V provides guidance for hypervariable region residues that can be changed. During antigen binding Hypervariable region residues or FR residues are generally used in a relatively conservative manner. Is replaced by Such conservative substitutions are listed in Table I under the heading "Preferred Substitutions". It was shown to. If such a substitution results in a change in biological activity If further shown in Table I for “typical substitutions” or for amino acid classes More essential changes are introduced and the product is screened as described below.                                   Table I  Substantial modifications in the biological properties of the antibody include (a) The structure of the polypeptide backbone in the area of its replacement, such as the cal tissue , (B) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. In selecting substitutions that differ significantly in their effect on Is achieved. Naturally occurring residues are grouped into groups based on customary side chain characteristics. Cracked:         (1) hydrophobicity: norleucine, met, ala, val, leu, ile;         (2) Neutral hydrophilicity: cys, ser, thr;         (3) acidic: asp, glu;         (4) bases: asn, gln, his, lys, arg;         (5) Residues affecting chain orientation: gly, pro; and         (6) aromatic; trp, tyr, phe.   Non-conservative substitutions exchange one member of these classes for another class It will be accompanied. Maintaining the proper structure of the humanized anti-CD11a antibody Any cysteine residues that are not included also improve the oxidative stability of the molecule. Can be generally replaced by serine to prevent and prevent abnormal cross-reactivity. You. Conversely, a cysteine bond can be added to an antibody to improve its stability (Especially when the antibody is an antibody fragment such as an Fv fragment).   Another type of amino acid variant of the antibody is the native glycosylation pattern of the antibody. To change. One or more carbohydrate moieties found in the antibody by alteration Delete and add one or more glycosylation that is not present in the antibody. Means   Glycosylation of antibodies is typically either N-linked or O-linked. N-linked involves the attachment of a carbohydrate moiety to the side chain of an asparagine residue. bird Peptide sequence asparagine-X-serine and asparagine-X-threonine (formula Where X is any amino acid other than proline) is a carbohydrate to the asparagine side chain It is a recognition sequence for enzymatic conjugation of the parts. Thus, this in the polypeptide The presence of any of these tripeptides creates a potential glycosylation site. O-linked glycosylation is 5-hydroxyproline or 5-hydroxylysine Although hydroxy amino acids, most commonly serine or threo, may be used Conjugation of sugar N-galactosamine, galactose, or xylose to nin .   The addition of glycosylation sites to the antibody can be caused by one or more of the above-mentioned tris. Amino acid sequences such as those containing a peptide sequence (for N-linked glycosylation sites) Advantageously achieved by changing the columns. The change also affects the original antibody distribution. Addition of one or more residues (for O-linked glycosylation sites) to the sequence Or by substitution therewith.   A nucleic acid molecule encoding an amino acid sequence variant of a humanized anti-CD11a antibody It is prepared by various methods well known in the art. These methods are limited. Separation from natural sources (in the case of naturally occurring amino acid sequence variants) ) Or oligonucleotide-mediated (or site-controlled) mutagenesis, PCR mutagenesis Preparation of humanized anti-CD11a antibody and cassette mutagenesis of mutant or non-mutant form Including preparation by development.   Usually, amino acid sequence variants of the humanized anti-CD11a antibody are either heavy or light chain. The amino acid sequence of the original humanized antibody (eg, as in SEQ ID NO: 2 or 5) and At least 75%, preferably at least 80%, more preferably (less At least 85%, more preferably at least 90%, most preferably at least Will also have an amino acid sequence with 95% amino acid sequence identity. this Sequence identity and homology reach maximum percent sequence identity, if necessary. And any conservative substitutions such as part of sequence identity (see Table I above) After aligning sequences and introducing gaps in order not to take into account The percentage of amino acid residues in the sequence candidate having identity with the humanized anti-CD11a residue Is defined here as a percentage. N-terminal, C-terminal, or internal extensions within the antibody sequence Those without large, deleted or inserted may affect sequence identity or homology. Should be composed.           (V) Screening for biological properties   Having the features identified herein as desired in a humanized anti-CD11a antibody Antibodies are screened for that.   For antibodies that bind to an epitope on CD11a bound by the antibody of interest (Eg, binding of MHM24 antibody to CD11a Locking it), Antibodies, A Laboratory Manual, Cold Spring Harbor L aboratory, Ed Harlow and David Lane (1988) A cross-blocking assay of proteins can be performed. Alternatively, for example, Champe et al. J. Biol. Chem. 270: 1388-1394 (1995). Can determine whether the antibody binds to the epitope of interest.   Antibody affinity (eg, for human CD11a or rhesus monkey CD11a) Either peripheral blood mononuclear cells or rhesus monkey leukocytes as described in the Examples below. And can be determined by bond saturation. This one to determine antibody affinity According to the method, lymphocytes or rhesus monkey leukocytes were collected in a volume of 170 μl per well. Added to the plate and the plate is incubated for 2 hours at room temperature. Inn After the incubation, the cells are harvested and washed 10 times. The sample then counts Be counted. Assignment of nanomolar saturation plots to 4-parameter curve fits Data that changed the form of the_d(app) value is determined Is done. A preferred humanized antibody is about 1 × 10^-7Up to about; preferably about 1 × 10^-8Ma More preferably about 1 × 10^-9Up to; most preferably about 2 × 10^-TenK Those that bind human CD11a with a d-value.   It also has beneficial anti-adhesion properties in the "keratinocyte monolayer adhesion assay" It is also desirable to select a humanized antibody having Suitable antibodies are up to about 250 nM. Preferably up to about 100 nM; more preferably up to about 1 nM and usually Prevents Jurkat Cell Adhesion to Human Epidermal Keratinocyte-Expressed ICAM-1 Most preferably those having an IC50 (nM) value of up to 0.5 nM. You. According to this assay, normal human epidermal keratinocytes are obtained from culture flasks. Removed, 5x10^FiveSuspended in lymphocyte assay medium at a concentration of viable cells / ml Is done. Aliquots of 0.1 μl / well were then placed in flat bottom 96 well plates. Overnight; in suitable wells, 100 units / well of interferon-gas Stimulated by addition of comma. Jurkat clone E6-1 cells are labeled Washed, 1x10⁶Resuspended in cells / ml, 500 ng at 4 ° C. for 30 minutes Incubate by initiating a two-fold serial dilution at / ml. Keratinocytes After removal of the medium from the monolayer, 0.1 ml / well of labeled cells was added and 1 Incubate at 37 ° C. for hours. The wells are used to remove unbound cells And the fluorescence is measured.   A preferred humanized anti-CD11a antibody is a mixed lymphocyte response using human lymphocytes. (MLR) assays up to about 100 nM; preferably up to about 50 nM More preferably an IC50 of up to about 5 nM, and most preferably up to about 1 nM. (nM) values. For both human and rhesus MLR, Peripheral blood from two unrelated donors was separated from heparinized whole blood and described below. In RPMI 1640 (GIBCO) with additives as described in the examples, 3x10⁶Resuspend to a concentration of cells / ml. The stimulator cells become unresponsive by irradiation. Responsiveness is created. 1.5x10 per well^FiveResponder cells at cell concentration Are co-cultured with an equal number of stimulator cells in a 96-well, flat bottom plate. A two-fold serial dilution starting at a concentration of 10 nM gives a total volume of 200 μl / well To be added to the culture. The culture is 5 days with 5% CO2_TwoMedium, at 37 ° C Incubated, then for 16 hours [^Three1 μCi / well of [H] thymidine Pulsed and [^Three[H] thymidine binding is measured.           (Vi) Antibody fragment   In one embodiment, the humanized anti-CD11a antibody is an antibody fragment . Various techniques have evolved for the production of antibody fragments. Traditionally, These fragments are derived via proteolytic digestion of whole antibodies (e.g., For example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-11 7 (1992) and Brennan et al., Science 229: 81 (1985)). However, these Fragments can now be produced directly by recombinant host cells. For example, F The ab'-SH fragment can be recovered directly from E. coli and F (ab ')_TwoChemically paired to form fragments (Carter et al., Bio / Technology  10: 163-167 (1992)). According to another approach, F (ab ')_TwoFragments are paired It can be isolated directly from the recombinant host cell culture. Other for the production of antibody fragments Methods will be apparent to the skilled expert.         (Vii) Multispecific antibody   In any embodiment, the binding for at least two different epitopes Generate multispecific (eg, bispecific) humanized anti-CD11a antibodies with mixed specificity May be desirable. An example of a bispecific antibody is the CD11a protein Can bind to two different epitopes. Alternatively, the anti-CD11a arm has a T Cell receptor molecules (eg, CD2 or CD3), or cells expressing CD11a FcγRI (CD64), FcγRII (CD32) for focusing defense mechanisms And Fc receptor for IgG (FcγR) such as FcγRIII (CD16) An arm that binds to the triggering molecule on leukocytes, such as, can be attached. Bispecific Antibodies can also be used to localize cytotoxic factors to cells expressing CD11a. Can be used. These antibodies have a CD11a binding arm and a cytotoxic factor (eg, Saporin, anti-interferon alpha, vinca alkaloid, ritin A chain, methotrex Xanthate or radioisotope hapten). Bispecific Antibodies can be full-length antibodies or antibody fragments (e.g., F (ab ')_TwoBispecific antibody).   According to another approach to making bispecific antibodies, the boundary between pairs of antibody molecules is Aspects minimize the percentage of heterodimers recovered from recombinant cell culture Can be processed to The preferred interface is the C of the antibody constant domain._H3 Contains at least part of the domain. In this method, the interface between the first antibody molecule One or more of these smaller amino acid side chains is replaced with a larger side chain (eg, Or tryptophan). Same or similar to the larger side chains The “cavities” that make up the size are smaller (for example, alanine or Replaces the large amino acid side chain with threonine) Created on the interface of molecules. This is another unwanted like homodimer Providing a mechanism for increasing the yield of heterodimers over terminal products You. See WO 96/27011 published September 6, 1996.   Bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, in the heteroconjugate antibody One antibody can bind to avidin, as well as biotin. Heteroconjugate antibodies It can be made using any suitable crosslinking method. Suitable crosslinkers are well known in the art And, along with a number of crosslinking techniques, are disclosed in U.S. Pat. No. 4,676,980.   Methods for generating bispecific antibodies from antibody fragments are also described in the literature Have been. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describes the procedure and describes a complete antibody Is F (ab ')_TwoProteolytic cleavage to produce fragments. these Fragments are used to stabilize nearby dithiols and to form intermolecular disulfides. Make To prevent it is reduced in the presence of the dithiol complexing agent sodium arsenate. The Fa The b 'fragment is converted to a thionitrobenzoate (TNB) derivative. F One of the ab'-TNB derivatives is obtained by reducing Facap by reduction with mercaptoethylamine. b'-thiol, which is further converted to another Fab'- to form a bispecific antibody. It is mixed with an equimolar amount of the TNB derivative. The bispecific antibody produced is selective for the enzyme. Can be used as an agent for proper immobilization.   Recent advances have focused on Escherichia coli, which can be chemically paired to form bispecific antibodies. They facilitate the direct recovery of their Fab'-SH fragments. Shalaby et al. E xp. Med. 175: 217-225 (1992) is a fully humanized bispecific antibody F (ab ')_TwoMolecular life Describe the birth. Each Fab 'fragment is independently secreted from E. coli. And in vitro directed chemical pairing to form bispecific antibodies? Be killed. The bispecific antibody thus formed is a human antibody against human breast tumor targets. Inducing the lytic activity of alveolar lymphocytes as well as HER2 receptor and normal It can be bound to cells that overexpress human T cells.   For making and isolating bispecific antibody fragments directly from recombinant cell culture Are also described. For example, bispecific antibodies are It is made using par. Kostelny et al. Immunol. 148 (5): 1547-1553 (199 2). Leucine zipper peptides from Fos and Jun proteins were In some cases, it bound to the Fab 'portion of a different antibody. The antibody homodimer is Reduced at the hinge region to form an antibody heterodimer. Is re-oxidized. This method can also be used for the production of antibody homodimers. You. Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) The "diabody" technique described is used to generate bispecific antibody fragments. Offers an alternative mechanism. The fragment is too short to Light chain variable domain by a linker that does not provide pairing between the two domains on the same chain (V_LHeavy chain variable domain (V_H)including. Therefore, one fragment V_HAnd V_LThe domain is the complementary V of another fragment_HAnd V_LPair with domain , Thereby forming two antigen-binding sites. Single chain F for generating bispecific antibody fragments by use of the v (sFv) dimer Another strategy Have also been reported. Gruber et al. Immunol. 152: 5368 (1994). Alternative In addition, the bispecific antibody is described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995) It may be referred to as "linear antibody" produced as described.   Antibodies with more than two valencies are contemplated. For example, a trispecific antibody is prepared. Tutt J. et al. Immunol. 147: 60 (1991).         (Viii) Other variants   Other variations of the humanized anti-CD11a antibody are contemplated. For example, in the treatment of cancer In order to increase the effectiveness of such antibodies, the antibodies of the invention may be used with respect to effector function. It may be desired to correct. For example, a cysteine residue is introduced into the Fc region. Thereby providing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capacity and / or It can increase complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Caron et al. Exp Med. 176: 1191-1195 (1992) and Shopes, B .; J. Immunol. 148: 2918-2922 (1992 ). Homodimeric antibodies with increased antitumor activity are described by Wolff et al., Callcer Re search 53: using a heterobifunctional crosslinker as described in 2560-2565 (1993) Can also be prepared. Alternatively, the antibody can be engineered to have two Fc regions. And thereby have increased complementing solubility and ADCC capacity. St See evenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).   The invention also relates to chemotherapeutic agents, poisons (e.g., bacteria, molds, plant or animal derived enzymes). Elementally active poisons, or fragments thereof), or radioisotopes (i.e., radioactive Immunoconjugate containing an antibody described herein conjugated to a cytotoxic agent such as Related. Chemotherapeutic agents utilized in the generation of such immunoconjugates are described above. Have been. Enzymatically active poisons and fragments thereof that can be used are diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain Domonas aeruginosa), ricin A chain, abrin A chain, modexin A chain , Alpha Sarsin, Aleurites fordii protein, Diantin protein, Ph ytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia Inhibitor, curcin, crotin, sapaonaria officinalis inhibitor ー, gelonin, Mitgelin, restrictocin, phenomycin, enomycin and tricosese Including The radionuclide of the nuclide is used for the production of radioconjugated anti-CD11a antibody. Available. An example is²¹²Bi,¹³¹I,¹³¹In,⁹⁰Y and¹⁸⁶Including Re.   The conjugate of the antibody and cytotoxic agent is N-succinimidyl-3- (2-pyridyldithi All) propionate (SPDP), iminothiolane (IT), imidoester Bifunctional derivatives (such as dimethyl adipimidate hydrochloride), active esters (disc Aldehydes (like glutaraldehyde), aldehydes (like cinimidyl suberate) , Bisazo compounds (bis- (p-azidobenzoyl) hexanediamine), bisdia Zonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine ), Diisocyanates (such as tolylene 2,6-diisocyanate), and bis -Like active fluorine compounds (like 1,5-difluoro-2,4-dinitrobenzene) It is produced using various bifunctional protein coupling agents. For example, Litin The immunotoxin was prepared as described in vitetta et al., Science 238: 1098 (1987). obtain. Carbon-14-labeled 1-isothiodianatobenzyl-3-methyldiethylenetria Minpentaacetic acid (MX-DTPA) is a reference for conjugation of radionucleotides to antibodies. It is a typical chelating agent. See WO 94/11026.   In another embodiment, the antibody is a “reagent” for use in tumor pre-targeting. May be conjugated to a sceptor (such as streptavidin), where the antibody- Scepter conjugates are used to remove unbound conjugates from the circulation using clarifiers and cytotoxicity Following administration of a “ligand” (eg, avidin) conjugated to the agent (eg, a radionuclide) Administered to patients.   The anti-CD11a antibodies disclosed herein can also be prepared as immunoliposomes. . Liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 368 8 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77: 4030 (1980); and US patents Nos. 4,485,045 and 4,544,545, by methods well known in the art. Prepared. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556. Disclosed within.   Particularly useful liposomes are phosphatidylcholine, cholesterol and PEG. A lipid composition comprising derivatized phosphatidylethanolamine (PEG-PE) When It can be produced primarily by the reversed-phase evaporation method. Liposomes are desirable Press through a defined pore size filter to obtain liposomes with a diameter. It is started. The Fab 'fragment of the antibody of the present invention can be used for disulfide alternating reaction. Via Martin et al. Biol. Chem. 257: 286-288 (1982). It can be conjugated to a posome. Chemotherapeutic agents (such as doxorubicin) May be arbitrarily contained in the inside of the. Gabizon et al. National Cancer Inst. 81 (19): See 1484 (1989).   The antibodies of the present invention convert prodrugs to active anticancer drugs (e.g., peptidylated (See WO 81/01145) Antibody to prodrug activating enzyme Can be used in ADEPT by combining them. For example, WO 88/073 No. 78 and U.S. Pat. No. 4,975,278.   The enzyme component of an immunoconjugate useful for ADEPT makes it more active, Act on prodrugs in such an approach to convert to cytotoxic forms Including any enzyme that can.   Enzymes useful in the method of the present invention include, without limitation, phosphoric acid Alkaline phosphatase useful for converting prodrugs containing free drugs into free drugs Arylsulfates useful for converting sulfate-containing prodrugs to free drugs Phatase; converts non-toxic 5-fluorocytosine to an anticancer drug, 5-fluorouracil Cytosine deaminase useful for converting; peptide-containing prodrugs as free drugs Seratia Protease, Sermolysin, Subtilis Useful for Converting to Of syn, carboxypeptidase and cathepsin (such as cathepsins B and L) Such proteases; useful for converting prodrugs containing D-amino acid substitutions D-alanyl carboxypeptidase: free glycosylated prodrug Carbohydrates such as .BETA.-galactosidase and neuraminidase useful for conversion into drugs Chloride-cleaving enzyme; β-lac useful for converting β-lactam derivatized drug to free drug By means of ctamases; and respectively phenoxyacetyl or phenylacetyl groups Useful for converting drugs derivatized at their amine nitrogen to free drugs. Penicillin amidases such as nicillin V amidase or penicillin G amidase Including ze. Alternatively, an enzyme, also known in the art as "abzyme", Have elementary activity Antibodies can be used to convert a prodrug of the invention to a free active drug. (See, for example, Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugation The body may be used as described herein for delivery of the abzyme to a tumor cell population. Can be prepared.   The enzymes of the present invention are useful in the art, such as the use of heterobifunctional crosslinking reagents described above. It can be covalently linked to the anti-CD11a antibody by well known methods. Alternatively, At least an antigen of an antibody of the invention bound to at least a functionally active portion of the light enzyme Fusion proteins with a binding region can be prepared using recombinant DNA techniques well known in the art. (See Neuberger et al., Nature 312: 604-608 (1984)).   In one embodiment of the present invention, complete anti-tumor activity, e.g., to increase tumor penetration It may be desirable to use antibody fragments rather than the body . In this case, the antibody fragment was added to increase its serum half-life. It may be desirable to make changes. This can be, for example, into antibody fragments. It can be achieved by the merging of salvage receptor binding epitopes (e.g., By mutation of the appropriate region in the body fragment or within the peptide tag. Antibody fragmentation, either by merging the tops, i.e., either terminally or moderately (Eg, by DNA or peptide synthesis). October 17, 1996 See published WO 96/32478.   Salvage receptor binding epitopes generally make up the region, where Fc Any one or more amino acids from one or more loops of the domain Residues are transferred to analogous positions in the antibody fragment. More preferably, Fc domain Three or more residues from one or two loops of the in are transferred. further More preferably, the epitope is the CH2 domain (eg, of an IgG) of the Fc region. CH1, CH3, or V_HArea, or one or more It is transferred to such an area. Alternatively, the epitope is the CH2 domain of the Fc region. Removed from the main and the C of the antibody fragment_LArea or V_LArea or both Is transferred to.   In a most preferred embodiment, the salvage receptor binding epitope is The sequence (5 ′ to 3 ′): PKNSSMISNTP (SEQ ID NO: 16) and HQSLGTQ (sequence Turn No .: 17), HQNLSDGK (SEQ ID NO: 18) and HQNISDGK (SEQ ID NO: 19), or The antibody fragment is Fab or F (ab ')_TwoVISSHLGQ (SEQ ID NO: 20) optionally further comprising a sequence selected from the group consisting of: Another most preferred In an embodiment, the salvage receptor binding epitope has the sequence (5 'to 3') : HQNLSDGK (SEQ ID NO: 18), HQNISDGK (SEQ ID NO: 19), or VISSHLGQ (sequence No. 20) and a polypeptide comprising the sequence: PKNSSMISNTP (SEQ ID NO: 16).   Covalent modifications of the humanized CD11a antibody are also included within the scope of the invention. So They can be obtained by chemical synthesis or, if applicable, the enzymatic or chemical Can be made by mechanical cutting. Other types of covalent modifications of the antibody were selected Antibody targeting by organic derivatizing agents capable of reacting with side chains or N- or C-terminal residues Is introduced into the molecule by the reaction of the functionalized amino acid residue.   Most commonly, the cysteinyl residue is a carboxymethyl or carboxamide An α-halide such as chloroacetic acid or chloroacetamide may be Reacted with roacetate (and the corresponding amine). Cysteinyl residues are also Lomotrifluoroacetone, α-bromo-β- (5-imidozoyl) propionic acid, Chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyldis Sulfide, methyl 2-pyridyl disulfide, p-chloromercury benzoate, 2 -Chloromercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxo Induced by reaction with sa-1,3-diazole.   Histidyl residues indicate that this agent is relatively specific for the histidyl side chain. It is induced by reaction with diethylpyrocarbonate at pH 5.5-7.0. Bromide Labromophenacyl is also useful; the reaction is 0.1 M cacodyl at pH 6.0. It is preferably carried out in sodium acid.   Lysinyl and amino terminal residues are reacted with succinic or other carboxylic anhydrides. Let Derivatization with these agents has the effect of reversing the charge of ricinyl residues . Another suitable reagent for derivatizing an α-amino containing residue is methylpicolin Imidate, pyridoxal phosphate, pyridoxal, borohydride trinitrate Robenzenesulfonic acid, O-methylisourea, 2,4-pentanedione Includes transaminase-catalyzed reactions with midesters and glyoxylates.   Arginyl residues include phenylglyoxal, 2,3-butanedione, Xandione, and one or several of the usual reagents of ninhydrin, with Is modified by the reaction of Derivatization of arginine residues is based on the guanidine function. High pKa requires that the reaction be performed in alkaline conditions . In addition, these reagents, like the arginine epsilon amino group, also have a lysine group. Can react with   The specific modification of the tyrosyl residue can be performed by using an aromatic diazonium compound or tetranitromethane. Features of introducing spectral labels within tyrosyl residues by reaction with tan Can be made with different interests. Most commonly, N-acetylimidizole and tet Lanitromethane forms O-acetyl tyrosyl species and 3-nitro derivatives, respectively Used to Tyrosyl residues are useful for use in radioimmunoassays. To prepare labeled proteins for¹²⁵I or¹³¹Iodized using I It is.   The carboxyl side chain (aspartyl or glutamyl) is 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azoni Carbodiimides such as (a-4,4-dimethylphenyl) carbodiimide (R--N = C = N -R ') wherein R and R' are different alkyl groups. Is done. In addition, aspartyl and glutamyl residues react with ammonium ions. Upon conversion to asparaginyl and glutaminyl residues.   Glutaminyl and asparaginyl residues have the corresponding glutamyl and asparagine residues, respectively. It is often deamidated to a partyl residue. These residues are neutral or basic conditions Deamidated under Deamidated forms of these residues are within the scope of the invention.   Other modifications include hydroxylation of proline and lysine, hydroxyl group of seryl or threonyl residues. Phosphorylation, methylation of α-amino group of lysine, arginine and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freema n & Co. , San Francisco, pp. 79-86 (1983)), acetylation of N-terminal amine, Amidation of the C-terminal carbonyl group.   Another type of covalent modification involves chemically or enzymatically coupling the antibody to the antibody. Including Kosyl. These methods are based on the fact that they can be used for N- or O-linked glycosylation. The fact that production of the antibody is not required in a host cell capable of glycosylation. Based on the coupling mode used, the sugar is (a) arginine And histidine, (b) free carboxyl groups, (c) free Such as those of the rufidryl group, (d) serine, threonine, or hydroxyproline. Such as free hydroxyl groups, (e) phenylalanine, tyrosine, or tryptophan Or (f) an amide group of glutamine. these The method is described in WO 87/05330 published September 11, 1987, and by Aplin and Wristo. n, CRC Crit. Rev. Biochem. , Pp. 259-306 (1981).   Removal of any carbohydrate moieties present on the antibody can be accomplished chemically or enzymatically. Can be achieved. Chemical deglycosylation involves the compound trifluoromethanesulfonic acid, Or requires exposure of the antibody to an equivalent compound. This treatment is based on the binding sugar (N-A Most or all except cetyl glucosamine or N-acetylgalactosamine) This results in the cleavage of the sugar, while leaving the antibody completely. Chemical deglycosylation is Hakimuddin et al., Arch. Biochem. Biophys. 259: 52 (1987) and Edge et al., Anal. . Biochem. , 118: 131 (1981). Carbohydrate moieties on the antibody Cuts are described in Thotakura et al., Meth. Enzymol. 138: 350 (1987) Can be achieved by the use of various endo- and exo-glycosidases.   Another type of covalent modification of the antibody is disclosed in U.S. Patent Nos. 4,640,835; 4,301,144; 4,670,417; 4,791,192 or 4,179,337 And various non-protein polymers such as polyethylene glycol, Binding the antigen to one of pyrene glycol or polyoxyalkylene. Prepare. B. Vectors, host cells, and recombination methods   The present invention also provides an isolated nucleic acid encoding a humanized anti-CD11a antibody, comprising the nucleic acid. The present invention also provides vectors and host cells to produce, and recombinant techniques for producing the antibodies. You.   For recombinant production of the antibody, the nucleic acid encoding it is isolated and further purified. Inserted into a replication vector for cloning (amplification of DNA) or expression. The DNA encoding the monoclonal antibody is immediately isolated and subjected to conventional methods (eg, Oligonucleotides that can specifically bind to genes encoding the heavy and light chains of the antibody (With a reotide probe). Many vectors available It is. Vector constructions generally include one or more of the following: Without limitation: signal sequence, origin of replication, one or more marker residues Genes, enhancer elements, promoters, and transcription termination sequences.         (I) Signal sequence element   The anti-CD11a antibody of the present invention can be produced not only directly and recombinantly, but also Or a signal sequence or other polypeptide which is a mature protein or polypeptide. Polypeptide with a heterologous polypeptide having a specific cleavage site at the N-terminus of the peptide May be produced as The heterologous signal sequence selected is recognized by the host cell. And processed (ie, with a signal peptidase). Native Prokaryotic host cells that do not recognize or process the anti-CD11a antibody signal sequence In the case of vesicles, this signal sequence is, for example, a prokaryotic signal sequence consisting of Selected from: alkaline phosphatase, penicillinase, lpp, or heat Qualitative enterotoxin II leader. For yeast secretion, the native signal sequence Are, for example, yeast invertase leader, α-factor leader (Saccharomyces ( Saccharomyces) and Kluyveromyces (Kluyveromy) es), acid phosphatase leader, C. ces). Albica Albicans glucoamylase leader, or WO 90/1364 6 may be substituted. In mammalian cell expression Indicates that the mammalian signal sequence is a viral secretory leader, such as herpes simplex gD. Available as well as signals.   Such a precursor region contains leading DNA in the DNA encoding the anti-CD11a antibody. Connected at the same time.         (Ii) Replication origin element   Both expression and cloning vectors contain one or more selected It contains a nucleic acid sequence that allows the vector to replicate within the host. Typically In cloning vector, this sequence is independent of the chromosomal DNA of the host. Duplicate And contains an origin of replication or an autonomous replication sequence . Such sequences are known for various bacteria, yeasts, and viruses. Step The origin of replication from Rasmid pBR322 is suitable for most Gram-negative bacteria and The μ plasmid origin is suitable for yeast and various viral origins (SV40, polio) Virus, adenovirus, VSV, or BPV) It is useful for a training vector. Generally, components of the replication origin are mammalian origins. The current vector (the SV40 origin may typically be used, but this is For the sole reason that it includes           (III) Selection gene element   Expression and cloning vectors may contain a selection gene or a selectable marker. Can be included. Typical selection genes include (a) antibiotics and other Toxins, such as ampicillin, neomycin, methotrexate, or Confer resistance to clin, (b) complement auxotrophic deficiencies, or (c) compare Complex media, such as the gene encoding D-alanine racemase against Bacillus subtilis Encodes a protein that supplies important nutrients not available from   One example of a selection scheme uses an agent to arrest growth of a host cell. Non Cells successfully transformed with the homologous gene produce proteins that confer drug resistance. And thus survive in the selection program. An example of such a dominant selection is neomycin Uses mycophenolic acid and hygromycin drugs I do.   Another example of a suitable selectable marker for mammalian cells is DHFR, thymidine kinase Ze, metallothionein-I and -II, preferably primate metallothionein Such as genes, adenosine deaminase, ornithine decarboxylase, etc. Enables the identification of competent cells that take up the CD11a antibody nucleic acid Things.   For example, cells transformed with a DHFR-selective gene may first be All transformants in a culture solution containing the tagonist methotrexate (Mtx) And identified by culturing. When using wild-type DHFR The host cell is a Chinese hamster ovary (CHO) with a deletion in DHFR activity. Cell line.   Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) can be 11a antibody, wild-type DHFR protein, and aminoglycoside 3'-phosphotran Transformation with a DNA sequence encoding another selectable marker such as spherase (APH) The transformed or co-transformed product is an aminoglycoside antibiotic such as In a culture solution containing a selective agent for a selectable marker of syn, neomycin or G418 Can be selected by growing cells. US Patent No. 4,96 See 5,199.   A suitable selection gene for use in yeast is the t gene present in the yeast plasmid YRp7. rp1 gene (Stinchcomb et al., Nature, 282: 39 ( 1979)). This trp1 gene lacks the ability to grow in tryptophan , A selection marker for a mutant strain of yeast, eg, ATCC No. 44076 or PEP4-1 Cars (Jones, Genetics, 85:12 (19 77)). The presence of the trp1 disorder in the yeast cell genome was then attributed to tryptophan. Provides an effective environment for detecting transformants that grow in the absence of DNA. same Thus, the Leu2-deleted yeast strain (ATCC 20,622 or 38,626) Complemented with a known plasmid carrying the Leu2 gene.   Furthermore, 1. The vector derived from the 6 μm circular plasmid pKD1 is Cruyvero. It can be used for transformation of Myces (Kluyveromyces) yeast. . Alternatively, an expression system for large-scale production of recombinant calf chymosin is described in K. et al. Lactis (Landis) (Van der Berg, Bio / Technology, 8: 135 (1990)). Kluybemais Industries Stable multicopy expression vector for secretion of mature recombinant human serum albumin Are also disclosed (Fleer et al., Bio / Technology, 9: 968-975 (1991)).         (Iv) Promoter element   Expression and cloning vectors are usually recognized by the host organism and contain anti-CD It contains a promoter operably linked to the 11a antibody nucleic acid. In prokaryotic hosts Suitable promoters to use include the phoA promoter, β-lactamer Enzyme and lactose promoter systems, alkaline phosphatase, tryptophan ( trp) promoter system and hybrid promoters such as the tac promoter, etc. Motor included. However, other known bacterial promoters are also suitable. You. Promoters used in bacterial systems also contain a D-encoding anti-CD11a antibody. Shine-Dalgarno operatively linked to NA. D. ) Contains an array .   Promoters are also known for eukaryotic cells. Virtually all eukaryotic genes Is an AT-rich located approximately 25-30 bases upstream of the site where transcription is initiated Region. Found 70 to 80 bases upstream of the transcription start site of many genes Another sequence that can be used is the CNCAAT region, where N is any nucleotide. Also means. At the 3 'end of many eukaryotic genes, the Bol A tail is inserted into the 3' coding sequence. There is an AATAAA sequence that can be a signal added to the 'end. Of these arrays All are properly inserted into eukaryotic expression vectors.   Examples of suitable promoter sequences for use in yeast hosts include 3-phosphoglycerate Nase or enolase, glyceraldehyde-3-phosphate dehydrogena Protease, hexokinase, birubic acid decarboxylase, phosphofructokiner Ze, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, Pyruvate kinase, triose phosphate isomerase, phosphoglucose Includes promoters for isomerase and other glycolytic enzymes such as glucokinase It is.   Other yeast promoters have the added advantage that transcription is regulated by growth conditions. Those that are inducible promoters with dots include alcohol dehydrogenase 2, Isocytochrome C, acid phosphatase, degradative enzymes involved in nitrogen metabolism, Tarothionine, glyceraldehyde-3-phosphate dehydrogenase, And the promoter region for enzymes involved in maltose and galactose utilization. is there. Suitable vectors and promoters for use in yeast expression are also E. P73,657. Yeast enhancers are also yeast promoters. -It is used advantageously.   Transcription of an anti-CD11a antibody from a vector in a mammalian host cell, for example, For example, polyoma virus, infectious epithelioma virus, adenovirus (eg, adenovirus) Virus 2), bovine papilloma virus, avian sarcoma virus, cytomegalo Ills, retrovirus, hepatitis B virus, and most preferably simian virus A promoter obtained from the genome of a virus such as irs40 (SV40), Heterologous mammalian promoters, such as actin promoters or immunoglobulins Promoters obtained from Blin promoters, and also heat shock promoters , Provided that such promoters are compatible with the host cell system. There is a condition that they are things.   The early and late promoters of the SV40 virus also contain the SV40 origin of replication It is conveniently obtained as an SV40 restriction fragment. The very early days of human cytomegalovirus The promoter is conveniently obtained as a HindIII E restriction fragment. mammalian DNA expressed using bovine papilloma virus as a vector One such system is disclosed in US Pat. No. 4,419,446. Modify this system No. 4,601,978. Herpes simplex Human β-Interface under the control of the thymidine kinase promoter from Irus Reyes et al. (Nature, 297: 598-601 (1982)). Alternatively, Rous sarcoma virus Terminal repeats can also be used as promoters.         (V) Enhancer element element   The DNA encoding the anti-CD11a antibody of the present invention is obtained from higher eukaryotes. Transcription is often increased by inserting an enhancer sequence into the vector. Known mammalian genes (globin, elastase, albumin; α-fetopro (Tein, and insulin), many enhancer sequences are known. Only Typically, however, as an example of using an enhancer from a eukaryotic cell virus Is the SV40 enhancer located on the rear side (100-270 bp) of the replication origin. Enhancer of the itomegalovirus early promoter, behind the origin of replication Eukaryotic promoters containing polyoma and adenovirus enhancers Yaniv's literature on Yinjin sequence elements that activate the promoter ( Nature, 297: 17-18 (1982)). Enha Is located 5 'or 3' to the sequence encoding the anti-CD11a antibody in the vector. Although they may be conjugated, they are preferably located 5 'to the promoter.         (Vi) transcription termination element   Eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or other multicellular organisms Expression vectors used in nucleated cells of origin also include termination of transcription and mRN. Sequences required for stabilization of A will be included. Such sequences are usually eukaryotic Or 5 ', sometimes 3' of the untranslated region of viral DNA or cDNA You can get more. These regions contain the mR encoding the anti-CD11a antibody. Nucleotide sequences transcribed as polyadenylation fragments in the untranslated region of NA Contains columns. One of the beneficial transcription termination elements is bovine growth hormone polyadenylate. Area. WO94 / 11026 and expression vectors disclosed therein Please refer to the         (Vii) Selection and transformation of host cells   In the present application, a vector for expressing or cloning DNA in a vector Suitable hosts include, as described above, prokaryotic cells, yeast, or higher eukaryotic cells. There is. Prokaryotic cells suitable for this purpose include gram-negative or gram-positive organisms. Such eubacteria, for example, Escherichia (eg, E. coli (E. coli) ), Enterobacter, Erwinia, Klebsi Ella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (eg : Salmonella. Salmonella typhimurium, Serratia (S erratia) (eg, Serratia marcescans), and Bacilli, as well as Enterobacteriaceae such as Shigella (Example: B. Subtilis, B.I. Licheniformis (E.g., B.B.I. disclosed in DD 266,710 issued on Apr. 12, 1989). Licheniformis (41P)), Pseudomon as) (Example: P. Aeruginosa) and Streptomyces (Streptom) yces). Other strains, such as E. coli B, E. coli. coli X177 6 (ATCC 31, 537); coliW3110 (ATCC27,3 25) is also suitable, but one of the preferred E. coli cloning hosts is , E .; coli 294 (ATCC 31, 446). These are not limiting, but exemplify Is what you do.   In addition to prokaryotic cells, eukaryotic microbes such as filamentous fungi or yeast also have anti-CD Suitable as a host for cloning or expression of a vector encoding the 11a antibody . Saccharomyce cerevisiaes or baker Known as yeast strains, they are the most commonly used of lower eukaryotic host microorganisms. It is. However, many other genera, species, and strains are available and Are also beneficial, for example, Schizosaccharomyces pom be); Kluyveromyces host (eg, K. Lactis , K .; Fragilis (ATCC 12,424); Bulgarix ( bulgaricus) (ATCC 16,045); Wickeramii (A) TCC 24, 178); Waltii (ATCC 56,500), K . Drosopilarum (ATCC 36, 906); Thermoto ・ Relance (thermotolerans) and K.K. Marxianus); Yarrowia (EP402,226); Pichia pastoris (Pichia pastoris) (EP183,070); Candida; Trichoderma Esia (Trichoderma reesia) (EP244, 234); Neurosporaq Lassa (Neurospora crassa); Schwanniomyces (example) : Schwanniomyces occidentalis ); And filamentous fungi (eg, Neurospora, Penicillium) llium), Trypocladium, and Aspergis llus) host (eg, A. Nidulans, and A. Niger) is there.   Suitable host cells for expressing glucosylated anti-CD11a antibodies are Derived from spore organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants, and Spodoptera frugiperda (Spo doptera frugiperda (caterpillar), Aedes aegypti (Aedes aegypti) ) (F), Aedes albopictus (f), Drosov Drosophila melanogaster (Drosophila), and Corresponding permissive insect hosts from hosts such as Bombyx mori cell Have been identified. Numerous transfection virus strains such as autographa L-1 mutant of Refornica (Autographa californica) NPV and Bombi Bm-5 strain of Bombyx mori NPV is available and such According to the present invention, a virus can be used as the virus of the present application, Especially for transfection of Spodoptera frugiperda cells Can be used for   Cotton, corn, potato, soybean, petunia, tomato, and tobacco Can be used as a host.   However, cells of spinal cord organisms are of most interest, Propagation by culture (tissue culture) has become a routine method. Beneficial mammalian host Examples of cells include monkey kidney CV-1 strain (COS-7) transformed with SV40. ATCC CRL1651); human embryonic kidney strain (293, or in suspension culture). A subclone of expanded 293 cells, Graham et al. Gen. Viro l. 36:59 (1977)) Baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Url aub et al., Proc. Natl. Acad. Sci. USA 77: 4216 ( 1980)); mouse Sertoli cells (TM4, Mather, Biol. Rep rod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATC) CCCL70); African green monkey kidney cells (VERO-76, ATCC C RL-1587); human cervical cancer cells (HELA, ATCC CCL2); dog kidney Spleen cells (MDCK, ATCC CCL34); buffalo rat liver cells (BR L3A, ATCC CRL 1442); human lung cells (W138, ATCC C CL75); human liver cells (HepG2, HB8065); mouse mammary adenocarcinoma (MM T060562, ATCC CCL51); TRI cells (Mather et al., An) nal. N. Y. Acad. Sci. 383: 44-68 (1982)); MR C5 cells; FS4 cells; and a human hepatoma cell line (HepG2).   The host cell is an expression or cloning vector for producing the anti-CD11a antibody. To induce a promoter, select transformants, or In a normal nutrient medium modified appropriately to amplify the gene encoding the sequence Cultured in         (Viii) Culture of host cells   The host cells used to produce the anti-CD11a antibodies of the invention may be in various media. Can be cultured. Commercially available media such as Ham F10 (Sigma ), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium ((DMEM), Sigma) etc. cultures the host cells Suitable to do. Further, Ham et al. (Meth. Enz. 58:44 (197 9)), Barnes et al. (Anal. Biochem. 102: 255 (198 0)), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927, 762; 4,560,655; or 5,122,469; WO90 / 03Z 130; WO 87/00195; or as described in U.S. Patent No. Re 30,985. Any of these can be used as a medium for the host cells. These cultures Hormone and / or other growth factors (eg insulin , Transferrin, or epidermal growth factor), salts (eg, sodium chloride, Lucium, magnesium, and phosphate), buffers (eg, HEPES), Nuku Leotide (eg, adenosine and thymidine), antibiotics (eg, gentamicin) (Trademark) drug), trace elements (absent normally at final concentrations in the micromolar range). Supplemented with glucose or an equivalent energy source. You may. Any of the other necessary supplements known to those skilled in the art, at the appropriate concentration Can be included. Culture conditions such as temperature and pH depend on the host cells selected for expression. Used previously and will be apparent to those skilled in the art.         (Ix) Purification of anti-CD11a antibody   Using recombinant techniques, the antibody can be cultured intracellularly, in the periplasmic space, or directly. It can be produced so as to be secreted into the ground. Producing antibodies intracellularly If so, the first step is to remove particulate debris, i.e. host cells or lysed fragments, e.g. Remove by centrifugation or ultracentrifugation. Carter et al. Are secreted into the periplasmic space of E. coli A method for isolating antibodies is described (Bio / Technology 10: 163-). 167 (1992)). Briefly, the cell paste was washed with sodium acetate (pH 3 . 5) in the presence of EDTA and phenylmethylsulfonyl fluoride (PMSF) so Thaw for about 30 minutes. Cell debris can be removed by centrifugation. Antibodies are secreted into the medium In some cases, supernatants of such expression systems are generally first prepared from commercially available proteins. White matter concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units Use and concentrate. Protease inhibitors such as PMSF for any subsequent steps Can inhibit proteolysis and contain foreign contaminants, including antibiotics Quality growth may be prevented.   The antibody composition prepared from the cells is, for example, hydroxyapatite chromatograph. Utilize fee, gel electrophoresis, dialysis, and affinity chromatography And affinity chromatography is preferred. It is a refining technology. The suitability of Protein A as an affinity ligand is Depends on the Fc region species and isotype of any immunoglobulin present in the antibody. Exist. Using protein A to base human γ1, γ2, or γ4 heavy chains Antibodies can be purified (Lindmark et al., J. Am. Immunol. M eth. 62: 1-13 (1983)). All mouse isotypes and human For γ3, protein G is recommended (Guss et al., EMBOJ. 5 : 15671575 (1986)). Matrix with affinity ligand The matrix is often: agarose, but may be other matrices. machine Stable matrices such as controlled pore glass or poly (styrenedivinyl) Nyl) benzene, etc., have higher flow rates and shorter times than those performed on agarose. Process time. Antibody is C_HIf you have three regions, Bakerbon dABX ™ resin (JT Baker, Phillipsburg, N J) is useful for purification. Other techniques for protein purification, such as ion exchange columns, Tanol precipitation, reverse phase HPLC, chromatography on silica, anion or Is heparin SEP on cation exchange resin (eg polyaspartic acid column) HAROSE ™ chromatography, chromatofocusing, SDS- Fractionation by PAGE, ammonium sulfate precipitation, etc. is also possible depending on the antibody to be recovered. .   Following the preliminary purification step, a mixture comprising the antibody of interest and a contaminant With an elution buffer having a pH between 2.5 and 4.5, preferably at a low salt concentration (eg, about Low pH Hydrophobic Interaction Chromatograph for Use at 0-0.25M Salt) I put it on.   C. Formulation   Antibodies of the desired degree of purification and, where appropriate, a physiologically acceptable carrier, excipient, or For storage, in the form of a lyophilized formulation or aqueous solution by mixing a stabilizing agent A therapeutic antibody formulation (Remington's Pharmaceutical) ical Sciences, 16th edition, Osol, A .; Edit, (1980)). Acceptable carriers, excipients, or stabilizers are used at the dosages and concentrations employed. It is not toxic to the consumer and is not phosphate, citrate, or other organic Buffers such as acids; antioxidants containing ascorbic acid and methionine; preservatives (eg For example, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; Benzalkonium chloride, benzethonium chloride; phenol, butyl or ben Zyl alcohol: alkyl paraben such as methyl or propyl paraben; Cole; resorcinol; cyclohexanol; 3-pentanoe M-cresol); low molecular weight (less than about 10 residues) polypeptide; serum Proteins such as albumin, gelatin or immunoglobulin; polyvinylpyrrolidone Hydrophilic polymers such as glycine, glutamine, asparagine, histidine, and al Amino acids such as ginine and lysine; monosaccharides, disaccharides, Course, mannose, or other carbohydrates containing dextrin; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt forming counterions such as chromium; metal complexes (eg, Zn-protein complexes); and / or T WEEN ™, PLURONICS ™, or polyethylene glycol (PEG) and other nonionic surfactants.   The formulation herein may also include more than one active compound required for the particular indication being treated , Preferably those having complementary activities that do not adversely affect each other. You may go out. For example, it may be desirable to further provide an immunosuppressant Absent. Such molecules are suitably combined in amounts that are effective for the intended purpose. Let it exist.   The active ingredient may also be prepared by, for example, coacervation or interfacial polymerization. They may be incorporated into black capsules, but in this case, In a rug delivery system (eg, liposomes, albumin microparticles, microemulsions) Emulsion, nano-particles, and nanocapsules) or macroemulsion. Hydroxygel cellulose or gelatin-microcapsules and poly -(Methyl methacrylate) microcapsules. Such technology is called Re Mington's Pharmaceutical Sciences, 16 Edition, Osol, A .; Edited, (1980).   Formulations for use in in vivo administration must be sterile. This is a sterile filtration membrane Achieved immediately by filtration through.   Sustained-release preparations may be prepared. Suitable examples of sustained release include solid hydrophobic Include semi-permeable matrices containing antibodies made of conductive polymers, such as It takes the form of shaped articles such as films and microcapsules, for example. Lasting Examples of release matrices include polyesters, hydrogels (e.g., poly (2-hydr) Roxyethyl-methacrylate) or poly (vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L-glu Tamic acid copolymer, non-degradable ethylene vinyl acetate, degradable lactic acid-glyco Copolymers such as Lupron Depot ™ (lactic acid-glycol) Injectable microparticles comprising an acid copolymer and leuprolide acetate))), and poly-D -(-)-3-hydroxybutyric acid is included. Ethylene-vinyl acetate and milk Polymers such as acid-glycolic acid enable release of molecules for over 100 days In contrast, some hydrogels release proteins for shorter periods of time. Cap If cellular antibody remains in the body for a long period of time, as a result of exposure to moisture at 37 ° C They denature or aggregate, resulting in loss of biological activity and altered immunogenicity May occur. A rational strategy to stabilize according to the mechanism involved was invented sell. For example, the mechanism of aggregation is the formation of intermolecular SS bonds by thio-disulfide exchange. If it is discovered that it is forming, modify sulfhydryl residues or use acidic solutions. Freeze-drying, adjusting the water content, using appropriate additives, Stability can be achieved by developing a polymer matrix composition. is there.   D. Non-therapeutic use of antibodies   The antibodies of the present invention can be used as affinity purification vehicles. This In this method, the solid phase such as Sephadex resin or filter paper The body is immobilized using methods well known in the art. Immobilized antibody Is contacted with a sample for purification containing the CD11a protein (or a fragment thereof). The support is made of a suitable material that removes substantially all substances other than the CD11a protein in the sample. After washing with a solvent, CD11a is bound to the immobilized antibody. Finally support The body may be exposed to other suitable solvents that release CD11a from the antibody, such as glycine buffer. , PH 5.0.   Anti-CD11a antibodies may also be used in diagnostic assays for the CD11a protein, such as its generation. Could be useful in detecting the current in special cells, tissues, or serum is there.   In diagnostic applications, the antibody will be labeled with a detectable moiety. Less than Numerous labels are available that can be classified into the following categories:     (A)³⁵S,¹⁴C,¹²⁵I,^ThreeH, and¹³¹Radioisotopes such as I. For example Current Protocol in Immunology (Vol. 1, 2 Vol., Edited by Colligen et al., Wiley-Interscience, New York, New York, Pubs. (1991)). Thus, antibodies can be labeled with radioisotopes, and radioactivity can be Can be measured using a counter.   (B) fluorescent labels such as rare earth chelates (eg europium chelates) or Fluorescein and its derivatives, Rhodamine and its derivatives, Dansyl, Lissami , Phycoerythrin, and Texas Red are available. Fluorescent labels For example, the above Current Protocol in Immuncolo gy can be conjugated to an antibody. Fluorescence is firefly It can be quantified using a light meter.   (C) Various enzyme-substrate labels are available and are described in U.S. Patent No. 4,275,1. 49 provides reviews on some of these. This enzyme is commonly used Catalyze chemical changes in chromogenic substrates that can be measured by various techniques . For example, the enzyme may catalyze a color change in the substrate, which is measured spectrophotometrically. It is possible to specify. Alternatively, the enzyme alters the fluorescence or chemiluminescence of the substrate. You may let it. Techniques for quantifying the change in fluorescence are described above. Chemiluminescent substrates Electrically excited by a chemical reaction and then measurable (eg with a chemiluminescence meter) Or emit energy to the fluorescent receptor. Examples of enzyme labels include Luciferase (eg, firefly luciferase, bacterial luciferase; (License number 4,737,456), luciferin, 2,3-dihydrophthalazinedio , Malate dehydrogenase, urease, horseradish peroxida Such as peroxidase such as HRPO, alkaline phosphatase, β- Lactosidase, glucoamylase, lysozyme, saccharide oxidase (eg For example, glucose oxidase, galactose oxidase, and glucose-6 -Phosphate dehydrogenase), heterocyclic oxidase (eg, Urica And xanthine oxidase), lactoperoxidase, microperoxy And the like. Techniques for coupling enzymes to antibodies are described by O'Sullivan et al. For the preparation of enzyme-antibody conjugates for use in enzyme immunoassays of (Methods in Enzym. Edited by J. Langone & H. Va n Vunakis; Academic Press, New York, 73 147-1616 (1981)).   Examples of enzyme-substrate combinations include, for example:   (I) Horseradish peroxidase (HRPO) and peracid as substrate Hydrogen peroxide; wherein hydrogen peroxide is a dye precursor (for example, orthophenylenediamine ( OPD) or 3,3 ', 5,5'-tetramethylbenzidine hydrochloride ( Oxidizes TMB));   (Ii) Alkaline phosphatase (AP) and Parani as chromogenic substrate Trophenyl phosphate; and   (Iii): β-D-galactosidase (β-D-Gal) and chromogenic substrate (Eg, p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase (4-methylumbell iferyl-β-D-galactosidase).   Numerous other enzyme-substrate combinations are also available to those skilled in the art. these A general review includes U.S. Patent Nos. 4,275,149 and 4,318,9. See 80.   Sometimes the label is conjugated indirectly to the antibody. The skilled person has various skills to achieve this. You will know the art. For example, antibodies are available for biotin and the labels Avidin can be conjugated to any of the three broad categories. It is possible, and vice versa. Biotin selectively binds to avidin, thus The label can be conjugated to the antibody by this indirect method. Or In order to achieve indirect conjugation between the label and the antibody, the antibody is converted to a small hapten (eg, Digoxin) and one of the different types of labels described above is anti- Conjugate to a hapten antibody (eg, anti-digoxin antibody). Therefore, with the labeled antibody Is achieved indirectly.   In another aspect of the invention, the anti-CD11a antibody need not be labeled, Can be detected using a labeled antibody that binds to the CD11a antibody It is.   The antibodies of the present invention can be prepared by any known assay method, such as competitive binding assays, Use in contact and indirect sandwich and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Ma natural of Technologies, pp. 147-158 (CRC Pre ss, Inc. 1987)).   Competitive binding assays use labeled standards for binding to limited amounts of antibody. Quality depends on the ability to compete with the test sample analyte. CD11a in the test sample The amount is inversely proportional to the amount of standard that binds to the antibody. Determine the amount of standard to bind Antibodies are generally insolubilized before or after competition to facilitate The reference material and the analyte bound to the Provide convenient separation.   The sandwich assay uses two antibodies, each with the protein to be detected. It can be linked to immunogenic portions or epitopes of different quality. Sandwich In a bioassay, the test sample analyte is a primary antibody immobilized on a solid support. Bound by the body, after which the second antibody binds to the analyte, and thus becomes insoluble Form a complex. See, for example, U.S. Patent No. 4,376,110. No. The two antibodies may themselves be detectable and labeled (direct sandwich Assay) or using an anti-immunoglobulin antibody labeled with a detectable moiety It is also possible (indirect sandwich assay). For example, a sandwich One type of assay is an ELISA assay, where a detectable Part is an enzyme. In the case of immunohistochemistry, the tumor sample may be fresh or Frozen or embedded in paraffin, for example, formalin May be fixed by a preservative such as   Antibodies can also be used in in vivo diagnostic assays. Typically, Antibodies are radionuclides (eg,¹¹¹In,⁹⁹Tc,¹⁴C,¹³¹I,¹²⁵I,^ThreeH,³²P, Or³⁵S) and the location of the tumor using immunoscintigraphy It can be attached.   E. FIG. Diagnostic kit   As a matter of convenience, the antibodies of the present invention require a kit, i.e., a predetermined amount of reagents. Packaged together with instructions for performing the diagnostic assay. Can be provided. If the antibody is labeled with an enzyme, Will include the substrates and cofactors required by the enzyme (eg, detection A substrate precursor that provides a possible chromophore or fluorophore). In addition, stabilizers and buffers ( It is possible to include other additives such as eg block buffer or lysis buffer). . Substantially optimize assay sensitivity by greatly varying the relative amounts of various reagents Specify the concentration in the solution for the reagent to be used. In particular, the reagents should Containing the excipients that provide the desired reagent solution, usually a lyophilized dry powder. I can do it.   F. Therapeutic uses for antibodies   The anti-CD11a antibody of the present invention can be used for various LFA-1-mediated diseases described in the present application. It has been considered so that it can be used to treat.   The anti-CD11a antibody can be administered by any suitable means, This includes parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, but In the case of effective immunosuppressive treatment, intralesional administration (perfusion application or (Including contacting the graft with the antibody prior to transplantation). Parenteral note Entry includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Furthermore, The anti-CD11a antibody is suitably administered by pulse infusion, particularly to reduce antibody Do it. Preferably, dosing is by injection, most preferably intravenous or subcutaneous Injections, which may be medications, short-term or chronic More dependent.   In the case of disease prevention or treatment, the appropriate dosage of antibody will depend on the type of disease being treated, Severity and history of the disease, as defined in, whether administered for prophylactic or therapeutic purposes Or prior treatment, the patient's clinical history, and response to antibodies, and It will also depend on the prudent care of a doctor. Antibodies are suitably administered once or in series. Administered to the patient throughout the treatment.   Depending on the type and severity of the disease, for example, one or more separate administrations Thus, or by continuous infusion, about 1 μg / kg to 15 mg / kg ( For example, 0.1 to 2.0 mg / kg) of antibody may be It is a candidate. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg. In the kg range or higher, this depends on factors such as those mentioned above. a few days If the drug is administered repeatedly for a longer period of time, Treatment is sustained until well controlled. However, other medications may be beneficial Maybe. Treatment progress is easily monitored by conventional techniques and assays. It is. An exemplary dosing regimen is disclosed in WO 94/04188.   Antibody compositions are formulated, dosed, and administered according to the desired medical practice. . Factors to consider in this regard include the particular disease being treated, the particular mammal being treated. Type, clinical condition of individual patient, cause of disease, delivery site of drug, administration method, The schedule of administration and other factors known to the medical practitioner are included. Administer The "therapeutically effective amount" of an antibody will depend on such considerations, It also prevents, ameliorates or treats LFA-1-mediated diseases (rheumatism). Sex Treatment of arthritis, reduction of inflammatory response, induction of tolerance of immunostimulants, host transplantation Prevent immune reactions that may result in rejection or vice versa, or (Including stretching)). Such an amount is preferred Or toxic to the host or more significantly sensitizes the host to infection Lower than the amount.   Antibodies do not have to be, but they can help prevent disease in question. Is suitably formulated with one or more agents currently used for treatment. For example, in rheumatoid arthritis, antibodies are combined with glucocorticosteroids. Given together. In addition, T cell receptor peptide therapy should Adjunct therapy to prevent clinical signs of epidemic encephalomyelitis. Graft In some cases, the antibody is combined with an immunosuppressant, such as cyclosporin A, as defined above. Administered together or separately to modulate the immunosuppressive effect. Or, Or, additionally, a VLA-4 antagonist or other LFA-1 antagonis May be administered to a mammal suffering from an LFA-1-mediated disease. Other like this The effective amount of the drug will depend on the amount of anti-CD11a antibody present in the formulation, the disease to be treated or Depends on the type of treatment and other factors discussed above. These are generally the same Used in the dosage form and by the route of administration as used above, or Used at about 1-99% of the given dosage.   G. FIG. Product   In another aspect of the present invention, the composition comprises a substance that is useful for treating the above-mentioned diseases. An article of manufacture is provided. The product comprises a container and a label. Proper content Instruments include, for example, bottles, vials, syringes, and test tubes. This container And may be formed from various substances such as glass and plastic. This container Holds a composition that is effective in treating the condition and also provides a sterile access port. (E.g., container can be pierced with a needle for subcutaneous tissue injection) An intravenous solution bag or vial having a stopper). In the composition The active ingredient is an anti-CD11a antibody. Labels on or attached to containers Indicates that the composition is used for treating the condition of choice. The product further second A pharmaceutically acceptable buffer, such as phosphate buffered saline, Including those containing Ringer's solution and dextrose solution You may have. Furthermore, other substances that are desirable from a commercial or user standpoint And may include other buffers, diluents, filters, needles, And package inserts containing instructions for use.         Example   Production of humanized anti-CD11a antibody   This example demonstrates that a mouse anti-human CD11a monoclonal antibody, MHM24 ( Hildreth et al., Eur. J. Immunol. 13: 202-208 (1 983)), and describes the biological efficacy in vitro. of mouse In previous studies on MHM24, like other anti-CD11a antibodies, Have been shown to inhibit T cell function (Hildreth et al., J. Immu. nol. 134: 3272-3280 (1985); Dougherty et al., E ur. J. Immunol. 17: 943-947 (1987)). Mouse And humanized MAbs both bind human T cells to human keratinocytes. , Which is a model for the effective response of MHC class II antigens Prevents proliferation of T cells in response to non-self lymphocytes in the lymphocyte response (MLR) (McCabe et al., Cellular Immunol. 150: 364-). 375 (1993)). However, the mouse (Reimann et al., Cyto metry, 17: 102-108 (1994)), and humanized MAbs Except for chimpanzee CD11a, it is cross-reactive with nonhuman primate CD11a. Did not respond. Obtaining humanized MAbs useful for preclinical studies in rhesus monkeys In CDR-H2, which is one of the complementarity determining regions in the variable heavy chain region, Human binding to rhesus monkey CD11a by replacing four residues The converted MAb was re-manipulated. Cloning of the Rhesus monkey CD11aI-region And molecular modeling were performed on rhesus monkey C for lysine residues in the human CD11aI-region. Changes to a glutamine residue in the D11aI-region were detected in mouse and humanized MA. This suggested that bs did not bind to rhesus monkey CD11a.   Materials and methods   (A) Construction of humanized F (ab ') s   Mouse anti-human CD11a MAb, MHM24 (Hildreth et al., Eu r. J. Immunol. 13: 202-208 (1983); h et al. Immunol. 134: 3272-3280 (1985)) And sequenced. Useful for mutagenesis and also in E. coli Phagemid pEMX1 to obtain a plasmid for expressing F (ab) Was built. pB0475 (Cunningham et al., Science 243) 1330-1336 (1989)). 0, pEMX1 was humanized κ-subgroup I light chain and humanized A DNA fragment encoding a subgroup III heavy chain (VH-CH1); Contains the phosphatase promoter and the Shine-Dalgarno sequence, but Both of them are pAK, another pUC119-based plasmid previously described. 2 (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)). Unique Spel restriction site It is inserted between DNAs encoding the light chain and heavy chain of F (ab).   To construct the first F (ab) variant of humanized MHM24, site-specific Mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA)  82: 488 (1985)) with pEMX1 containing deoxyuridine. Performed on template; all six CDRs were changed to MHM24 sequence. other All of the F (ab) variants were constructed from the F (ab) -1 template. Double strand and one To prepare the single-stranded DNA, the plasmid was transformed into the E. coli strain XL-1 Blue (St ratagene, San Diego, CA). Each change For variants, use the dideoxynucleotide method for both light and heavy chains. And completely sequenced (Sequenase, US Biochemical Co.). rp. ). The plasmid was transformed into E. coli strain 16C9, a derivative of MM294. Then, spread on an LB plate containing 5 μg / ml carbenicillin, A single colony was selected for work. This single colony was 100 μg / ml The cells were grown at 37 ° C. for 5-8 hours in 5 ml of LB medium containing cillin. This 5m Culture of l The product was added to 500 ml of AP5-100 μg / ml carbenicillin and 4 liters were added. And grown in a baffled shake flask at 37 ° C. for 16 hours. APS medium is 1.5 g glucose, 11.0 Hycase SF, 0.6 g yeast extract ( Guaranteed), 0.19 g MgSO4 (anhydrous), 1.07 g NH4C1, 3.7 3 g KCl, 1.2 g NaCl 120 ml 1 M triethanolamine pH 7.4, added to 1 L of water and containing 0.1 μm Sealken filter. The solution was sterilized through a luter.   Cells are harvested by centrifugation at 3000 xg in IL centrifuge bottles (Nalgene) And the supernatant was removed. After 1 hour of freezing, the pellet was cooled to 25 ml in 10 m Resuspended in M MES-10 mM EDTA, pH 5.0 (buffer A). 2 50 μl of 0.1 M PMSF (Sigma) was added to inhibit proteolysis and 10 mg / Ml of hen egg white lysozyme stock at 3.5 ml / well to add to the bacterial cell wall. Assisted dissolution. After gently shaking on ice for 1 hour, the samples were weighed at 40,000 × g. Centrifuge for 15 minutes. The supernatant was made up to 50 ml with buffer A and equilibrated with buffer A 2 Loaded on ml DEAE column. The flow-through was adjusted to the protein equilibrated with buffer A. G-Sepharose CL-4B (Pharmacia) column (0.5 ml bed volume) Volume). The column was washed with 10 ml of buffer A and 3 ml of 0.3 M Eluted with Syn, pH 3.0 and placed in 1,25 ml 1 M Tris, pH 8.0. F (ab) is then buffered into PBS using Centricon-30 (Amicon) The solution was exchanged, and the final volume was concentrated to 0.5 ml. SDS for all F (ab) -Check the purity by running a PAGE gel and also by electrospray mass spectrometry The molecular weight of each variant was confirmed.   (B) Construction of chimeric and humanized IgG   A chimeric (chIgG1) human IgG1 variant and a humanized (HuIgG1 1) For MHM24, the appropriate mouse or humanized variable light and heavy chains ( F (ab) -8, Table II) regions, each described previously as a pRK vector. (Gorman et al., DNA Protein Eng. Tech. 2) : 3 (1990)). Of HuIgG1 light and heavy chains Site-directed mutagenesis of plasmids (Kunkel, Proc. tl. Acad. Sci. USA 82: 488 (1985)). Nin-scan mutants were constructed. The DNA sequence of each mutant was dideoxy Confirmed by nucleotide sequencing.   High efficiency method (Graham et al., J. Gen. Virol. 36:59 (1977) Gorman et al., Science, 221: 551 (1983)). The human fetal kidney transformed with the heavy and light chain plasmids by adenovirus 293 (Graham et al., J. Gen. Virol. 36:59 (1 977)). Replace the medium with serum-free medium for 5 days Collected. Using Protein A-Sepharose CL-4B (Pharmacia) Antibodies were purified from the pool of supernatants. The eluted antibody was used for Centricon-30 (A) The buffer solution was exchanged with PBS using Mikon), concentrated to 0.5 ml, and milled. Filtered with x-GV (Millipore) for sterilization and stored at 4 ° C.   Antibody concentrations were determined in a total Ig-binding ELISA. Humanized anti-p for reference 185^HERIgG1 (Carter et al., Proc. Natl. Acad. Sci. . USA 89: 4285 (1992)) determined by amino acid composition analysis. did. 1 μg / ml goat anti-human IgG was added to each well of a 96-well plate. F (ab ')_Two(Cappel Laboratories, Westches ter, PA) at 4 ° C. for 24 hours. Dilute the purified antibody and duplicate And added to the coated plate. After 1.5 hours incubation, play Washed with PBS-0.02% Tween20 and diluted 1: 2000 Sladish peroxidase conjugated sheep anti-human IgGF (ab ')_TwoAdd I got it. After a 1.5 hour incubation, the plate is washed and 0.1 ml of 0 . 2 mg / ml o-phenylenediamine dihydrochloride-0.01% peroxide Hydrogen-PBS was added. After 10 minutes, the reaction was stopped with 2M sulfuric acid and 490 nm 0. D. Was measured.   (C) Cloning of the rhesus monkey CD11aI-region   Using RT-PCR and primers derived from the human CD11a DNA sequence, The DNA sequence of the I region of Gesar was obtained. Simply put, Fast Track m Approximately 10 mRNA was prepared using an RNA purification kit (Invtorgen).⁷No Mosquito It was isolated from Gesar lymphocytes. 10 μg of mR using MuLV reverse transcriptase NA was reverse transcribed. The first-strand cDNA was then converted to 40 using the following primers: Amplified in cycles of PCR:   5'-CACTTTGGATACCGCGTCCTGCAGGT-3 '(forward) (SEQ ID NO: 21), and   5-CATCCTGGCAGGTCTGCCTTCAGGTCA-3 '(reverse direction ) (Sequence recognition number: 22).   Agarose gel electrophoresis shows a single band of the expected size from the PCR reaction. Purified. Digest the PCR product with the restriction enzyme Sse8387I (Takara) and It was ligated to a plasmid containing human CD11a digested with restriction enzymes. Human CD11a distribution There are two Sse8387I sites in the row, one on either side of the I-region. The resulting plasmid was human CD1 in which the human I-region was replaced with the rhesus monkey I-region. Encodes a chimera consisting of 1a. DNA sequence analysis shows that It was found that there was a difference of 5 amino acids between the two. One is the N-terminus of the I-region On the region side (Thr59Ser), the other four were within the I-region itself: Va1133Ile, Arg189Gln, Lys197G1u, and Va13 08A1a (FIG. 2).   (D) FACScan content of binding of F (ab) and IgG to Jurkat cells Analysis         For humanized and control antibodies, PBS-0.1% BSA-10m M serially diluted with sodium azide, 10⁶Jerkat T-cell Aliquots were incubated at 4 ° C. for 45 minutes. Wash cells, then Fluorescein conjugated goat anti-human F (ab ')_Two(Organon Tekni ka, Westchester, PA) at 4 ° C for 45 minutes. I did. The cells are washed and FACScan (Becton Dickinson, (Mountain View, CA). 8 × 10^ThreeCell list Gated with the front light scatter versus the side light scatter, Dead cells and debris were excluded.   (E) Apparent K_dSaturated bonds to determine s   Iodo-Gen (Pierce, Rockford, IL) Radiolabeled antibodies were prepared as indicated. 50 μg antibody and 1 m Ci's¹²⁵I (DuPont, Wilmington, DE) to each tube And incubated at 25 ° C. for 15 minutes. Radiolabeled protein The quality was determined using Hank's balanced salt solution containing 0.2% gelatin (HBSS, Life Te). (e.g., PD-Chnologies, Grand Island, NY). Using 10 columns (Pharmacia, Uppsala, Sweden), the remaining Free liberation¹²⁵Purified from I.   Lymphocyte separation medium (LSM, Organon Teknika, Durham , NC) were used according to the manufacturer's instructions and mononuclear cells were obtained from two donors. And purified from heparin-treated human peripheral blood. Blood is 25 at 400 xg for 40 minutes Centrifuge at 0 ° C without brake. Collect cells at the interface of LSM and plasma, then Resuspended in HBSS-0.2% gelatin.   Heparin treatment recovered from two rhesus monkeys by dextran precipitation Lymphocytes were purified from the peripheral blood. Blood is 3% dextra in an equal volume of PBS Diluted with T500 (Pharmacia) and left at 25 ° C. for 30 minutes to precipitate. . After the precipitation, the cells remaining in the supernatant are collected and centrifuged at 400 × g for 5 minutes to precipitate the cells. I let you go. The remaining erythrocytes were subjected to two cycles of distilled water and 2 × HBSS. Removed by hypotonic dissolution. After lysis of the red blood cells, the cells are washed with PBS and And resuspended in HBSS-0.2% gelatin.   The affinity of the antibody was determined for peripheral blood mononuclear cells (mouse MHM24 and HuIgG1), Indicates saturation using any of rhesus monkey lymphocytes (MHM23, RhIgG1). Determined by binding. In each assay, radiolabeled antibodies were Sets were serially diluted with HBSS-0.2% gelatin. Non-specific binding is best Serially dilute and add 500 nM final concentration of homologous unlabeled antibody in duplicate It was decided by that. Human or rhesus monkey lymphocytes per well Added to the plate in a volume of 170 μl. Plates are placed on an orbital plate shaker for 2 hours. Incubated for hours at room temperature. After incubation, SKATRON Harvest using a cell harvester (Lier, Norway), 0.25% Washed 10 times with PBS containing gelatin and 0.1% sodium azide. Next The sample was an LBK Wallac GammaMaster gamma counter. -(Gaitherburg, MD) for 1 minute. Data every minute From the counts of the above, it was converted to nanomolar concentration, and then Optimization (binding vs. total)_d(App) values were determined.   (F) Keratinocyte monolayer adhesion assay   Normal human epithelial keratinocytes (Clonetics, San Diego, CA) was removed from the culture flask with trypsin-EDTA, centrifuged, and lymphocytes Assay medium (RPMI1640 (GIBCO) -10% fetal calf serum-1% 5x10 in nisin / streptomycin)^FiveResuspend at a concentration of viable cells / ml did. An aliquot of 0.1 ml / well is then placed in a flat bottom 96-well plate. Cultured overnight; interferon-gamma (Genentech, South) San Francisco, CA) at 100 units / well and add the appropriate Stimulated the well.   Jurkat cell clone E6-1 (ATCC, Rockville, MD) Or purified rhesus monkey lymphocytes (see MLR method) at 20 μg / ml Calcein AM (Molecular Probe, Eugene, O.) R) for 45 minutes at 37 ° C. After washing three times with lymphocyte assay medium, 1 × 10 Jurkat or rhesus monkey lymphocyte cells⁶Resuspend in cells / ml, Incubated with serially diluted antibodies at 4 ° C. for 30 minutes. Kera After removing the medium from the monolayer of Tinocyte, 0.1 ml / well of labeled cells was added. And incubated at 37 ° C. for 1 hour. 0.2 ml / well / well Non-adherent cells were removed five times by washing with a lymphocyte medium at 37 ° C. five times. Cyto Measure fluorescence using fluor2330 (Millipore, Bedford, MA). Specified.   Rhesus monkey-human, comprising human CD11a with rhesus monkey I-region Chimeric CD11a (Rh / HuCD11a) was transformed by site-directed mutagenesis (Ku nkel, Proc. Natl. Acad. Sci. USA 82: 488 (1 985)), a deoxyuridine-containing template plasmid encoding human CD11a Constructed on Sumid. Four residues were changed: Va1133Ile, Arg1. 89Gln, Lys197G1u, and Va1308A1a (FIG. 2). RH / H Plasmids encoding uCD11bi and human CD11a (EP364,69) 0) by the high efficiency method (Graham et al., J. Gen. Virol. 36:59 (1) 977); Gorman et al., Science, 221: 551 (1983)). Utilizing an adenovirus-transformed human embryonic kidney cell line, 293 (Gr aham et al. Gen. Virol. 36:59 (1977)). Entered. 293 cells transfected with Rh / HuCD11a were transformed with 20 μg / ml Labeled with Calcein AM at 37 ° C for 45 minutes. In lymphocyte assay medium After three washes, 293 cells transfected with Rh / HuCD11a were added to 1 × 10⁶ Resuspend cells / ml and incubate with serially diluted antibody for 30 minutes at 4 ° C. It was cubified. After removing the medium from the keratinocyte monolayer, 0.1 ml / Wells of labeled 293 cells were added and incubated at 37 ° C for 1 hour. The wells were washed 5 times with 0.2 ml / well / times of lymphocyte medium at 37 ° C., Non-adherent cells were removed. Fluorescence was measured using Cytofluor 2300 .   (G) ICAM adhesion assay   Maxisorp (Nunc) 96-well plate with 0.1 ml / well of 1 μl g / ml goat anti-human IgG Fc (Caltag) for 1 hour at 37 ° C. Was. After washing the plate three times with PBS, the plate was washed with 1% BSA-PBS for 2 hours. Blocked at 5 ° C for 1 hour. The plate is then washed three times with PBS, 0.1 ml / Well of 50 ng / ml recombinant human ICAM-IgG and incubate overnight. Privation.   ICAM-IgG is 5 cells of human ICAM fused to human IgGFc It consists of an outer area. Human ICAM-1 (Simons et al., Cell, 331: 6 24-627 (1988), and Stauton et al., Cell, 52: 925-. 933 (1988)) is a plasmid for expression of an immunoadhesin, and contains pRK. 5d What was termed ICAMGaIG was constructed. This is the ICAM-1 Ig-like Five regions, Genenase I, an H64A mutant of subtilisin BPN ' (Carter et al., Proteins: Structure Function) , and Genetics 6: 240-248 (1989)). Cleavage sites of 6 amino acids and human IgG1 (Ellison et al., Nuclei c Acids Research 10: 4071-4079 (1982)) Of the Fc region of pRK5 vector (Eaton et al., Biochemistry 2). 5: 8343-8347 (1986)). Human fetal kidney 29 3 cells (Graham et al., J. Gen. Virol. 36:59 (1977)). ) With pRK. 5dICACAMGaIg and RSV-neo plasmid (Gorm , et al., Science, 221: 551-553 (1983)). A cell line expressing the five regions of ICAMIg (5dICAMIg) Created. Antibodies to human IgG Fc (Caltag, Burlingam) e, CA) and ICAM-1 (BBIG-II; R & D System, Min. neapolis, MN) using an enzyme-linked immunosorbent assay (ELISA). A clone expressing 20 μg / ml of secreted 5dICAMIg was selected from the above. . 0.01 M Hepes buffer (pH 7.0), 0.15 M NaCl (HB S), a ProteinA column (ProsepA, Bioproc) equilibrated with essing, Ltd. , Durham, England) cells of this cell line The culture supernatant is applied and the column is HBS, then 0.01M Hepes buffer (p H7.0), 0.5 M NaCl, 0.5 M TMAC (tetra-methyl ammonium) Um chloride) to remove non-specifically bound material. At HBS, TMAC The buffer was thoroughly washed from the column and 5d ICAMIg was added to 0.01 M Hepes. (PH 7.0) Dissolve in 3.5M MgCl2 and 10% (w / v) glycerol Issued. The Protein A pool was concentrated by extensive dialysis against HBS.   Purified rhesus monkey lymphocytes (see MLR method) l of Calcein AM (Molecular Probe, Eugene, O R) at 37 ° C. for 45 minutes. After washing three times with lymphocyte assay medium, 1 × 10 rhesus monkey lymphocyte cells⁶Cells resuspended in cells / ml and serially diluted Incubated with the body at 4 ° C. for 30 minutes. Coding with ICAM-IgG After removing the medium from the washed plate, 0.1 ml / well of labeled cells was added. And incubated at 37 ° C. for 1 hour. 0.2 ml / well / well so, The cells were washed 5 times with a lymphocyte medium at 37 ° C. to remove non-adherent cells. Cytoflu or2330 (Millipore, Bedford, Mass.) Was measured.   (H) One-way mixed lymphocyte response (MLR)   Lymphocyte separation medium (Organon T) was used for human and rhesus monkey MLR. eknika, Durham, NC) and two unrelated providers Was isolated from heparinized whole blood. Lymphocytes , RPMI 1640 (GIBCO) -10% human AB serum-1% glutamine-1 % Penicillin / streptomycin-1% non-essential amino acids-1% pyruvate 5 × 10^-FiveM 2-β-mercaptoethanol-50 μg / ml gentamicin 3 × 10 5 in -5 μg / ml polymyxin B⁶It was re-turbid at a concentration of cells / ml. Irradiate 3000 rads in a cesium irradiator to render the stimulator cells non-reactive. Was. 1.5 × 10 per well^FiveResponding cells at the same cell concentration as equal number of stimulating cells Originally, they were cultured together in a 96-well flat bottom plate. Dilute two times continuously Each antibody was added to the culture to a total volume of 200 μl / well. Culture Was cultured at 37 ° C. in 5% CO 2 for 5 days, and then 1 μCi / well of [^ThreeH] Chimi Pulsed with gin for 16 hours. [^ThreeH] thymidine incorporation, It was measured with an scintillation counter. Assays were performed in triplicate . Humanized anti-human p185^HERMAbs (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992)). And RhIgG1 were used as isotype controls. Mouse anti-hamster -TPA MAb (Genentech) was used as an eye to the MHM23 MAb Used as a sotype control (mouse IgG1). MAb 25.3 is Immu purchased from notech (Westbrook, ME).   (I) Computer graphics model for mouse and humanized MHM24   Using the sequences of the VL and VH regions (FIGS. 1A and B), mouse MHM24 A computer graphics model in the VL-VH domain was constructed. This model Was used to determine which framework residues to incorporate into the humanized antibody. F A model of (ab) -8 was also constructed to correct the selection of mouse framework residues. Was confirmed. The construction of the model was performed as previously described (Carter et al., Proc. Natl. Acad. Sci. USA 89: 4285 (1992); Eigenbrot et al. Mol. Biol. 229: 969 (1993)) went.         result   (A) Humanization   Consensus sequences of human heavy chain subgroup III and light chain subgroup κI Used as a framework for humanization (Kabat et al., Sequenceo) f Proteins of Immunological Interes. 5th Ed. Public Health Service, Nationa l Institute of Health, Bethesda, MD. (1 991)) (FIGS. 1A and B). Use this framework well for other mouse Was successfully humanized (Carter et al., Proc. Natl. Acad. Sci. ScL USA 89: 4285 (1992); Preta et al. Immun ol. 151: 2623-2632 (1993); Eigenbrot et al., Pr. oteins 18: 49-62 (1994)). First of all humanized variants Screening for F (ab) s expressed in Escherichia coli did. Typical yields are 0.2-0.500 per 500 ml shake flask. It was 5 mg of F (ab). By mass spectroscopy, each F It was confirmed that the mass of (ab) was within 5 mass units.   CDR-H1 contained residues H28-H35, as described in Kabat et al. Sequences of Proteins of Immunological al Interes. 5th Ed. Public Health Server ice, National Institute of Health, Bet hesda, MD. (1991)), and Chothia et al. (Nature, 3 42: 877-883 (1989)). Other super The variable loop was defined according to Chothia et al. (1989). Light chain residue number Is prefixed with L, and the number of residues in the heavy chain is prefixed with H.                                   Table II   Binding of humanized MHM24 mutant to human CD11a on Jurkat cells        a Residues in mouse are in bold; number of residues is in Kabat et al., Sequences   of Proteins of Immunological Inter st. 5th Ed. Public Health Service, Nati onal Institute of Health, Bethesda, MD . (1991).         b Mean and standard deviation are calculated for each independent FACscan analysis. EC50 for F (ab) -2 771 ± 320 ng / ml.         c HuIgG1 is fused to the constant region of human light and heavy chains by F (a b) -8 VL and VH regions.         d chIgGl is a mouse fusion of the constant regions of human light and heavy chains. Chimeric IgG with VL and VH regions.   In the first variant, F (ab) -1, the CDR residues are To the human framework. Furthermore, for residue H71, human Was changed from mouse Arg to mouse Val. And has been shown previously to affect the conformation of CDR-H2. (Chothia et al., Nature, 342: 877-883 (1989); T ramontano J .; Mol. Biol. 215: 175 (1990)). No detectable binding was seen with this F (ab). In F (ab) -2 Indicates that CDR-H2 has a sequence-based definition (ie, including residues H60-H65). Extended to include. EC5 for binding of F (ab) -2 to human CD11a 0 was 771 ± 320 ng / ml, which is the EC50 of chimeric IgG1. (5.2 ± 3.0 ng / ml).   In previous humanizations, the frames adjacent to CDR-H1 and CDR-H2 were Residues in the framework loop (FR-3) have been found to affect binding (Eigenbrot et al., Proteins 18: 49-62 (1994). ). In F (ab) -5, three residues in this loop correspond to the corresponding mouse And this variant showed a 23-fold improvement in binding ( Table II). When the residues of human L53 and L55 are changed to those of mouse (ie, Se rL53The, and GluL55Gln), a further 4 fold improved binding (F (Ab) -6, Table II); this defines CDR-L2 as structurally based (residue L5 0-L52) to an effective sequence-based definition (residues L50-L56). Was. Subsequent PheH27 in CDR-H1 to mouse Tyr A further 3-fold improvement was achieved with the conversion of (F (ab) -7; Table II). Finally 3 in FR-3, based on the mouse and humanized model of MHM24 Horn Two of the mouse residues (H75 and H76) were returned to humans and these two residues were linked. In this case, no effect was found (F (ab) -7 and F (ab)- 8; Table II). The average EC50 of F (ab) -8 is that of chimeric IgG Was slightly better (Table II). Not all human-to-mouse changes are binding It has not improved. Did you change PheH67 to mouse Ala? It was previously known that this site affects binding (Presta et al., J. Immunol. 151: 2623-632 (1993)), the effect is clear (F (ab) -3, Table II). ValH71 to human A Returning to rg reduced binding by a factor of 3 (F (ab) -4, Table II), Supports incorporation of Va1H71 into (ab) -1.   The VL and VH regions from F (ab) -8 were transferred to the constant region of human IgG1. Was. HuIgG1, a full-length intact antibody, has an EC5 equivalent to F (ab) -8. 0 and improved when compared to full length chimeric IgG1 (Table II) . Includes use as a standard for alanine-scan and MLR assays (hereinafter See below) Considering the data of all assays of HuIgG1, human CD11a EC50 of HuIgG against 0.042 ± 0.072 nM (N = 15) there were. A saturation binding assay was also performed to determine the apparent dissociation constant K_d(App) Determined: 0.15 ± 0.02 nM for mouse MHM24; HuIgG1 About 0.15 ± 0.04 nM (Table III).                                 Table III   Apparent K by saturation binding to human lymphocytes and rhesus monkey leukocytes_d         a Assay for rhesus monkey donor 3 is for RhIgG1 The assay was performed using 1 mg / ml human IgG. 1 in the presence of Fc receptor interaction.         (B) Alanine-scan of CDR residues         To determine which CDR residues are involved in binding to human CD11a, Alanine-scan for CDR residues of uIgG1 (Cunningham) Science, 244: 1081 (1989)). Each change Variants were tested for binding to CD11a on Jurkat cells. Light chain In this case, only CDR-L3 contributed to the binding. HisL91 has a great effect But (Table IV), this side chain must be partially buried, so Is probably a conformational one. Residues AsnL92 and TyrL94 are There was a modest effect, with a 3-fold and 12-fold reduction, respectively. However, These two residues were simultaneously changed to alanine (similar to GluL93A1a). Let Note that there is no additional effect on the coupling. Variant L3, Table IV).                                  Table IV       Alanine-scan for humanized MHM24 CDR residues                              Table IV (continued)        a CDR and FR-3 are defined in Kabat et al. (1991, supra). Things.         b EC50 of HuIgG1 against human CD11a = 0.042n M (SD = 0.072; N = 15).         c EC50 of HuIgG1 against rhesus monkey CD11a = 45. 6 nM (SD = 40.4; N = 16); total against Rhesus sarno CD11a All values are for a single binding assay unless otherwise noted; nb is Means binding of the mutant, more than 10 times weaker than HuIgG1.         d Multiple alanine variants:         H1, SerH28Ala / TheH30Ala / HisH32Ala;         H2, HisH52Ala / SerH53Ala / SerH55Ala;         H2B, AspH54Ala / GluH56Ala / ArgH58Ala;         H2A1, HisH52Ala / SerH53Ala;         H2A2, SerH53Ala / SerH55Ala;         H2A3, HisH52Ala / SerH55Ala;         H3, TryrH97Ala / TyrH99Ala;         H3B, ThrH100aAla / ThrH100bAla / TyrH100cAla;         L1, LysL27Ala / ThrL28Ala / SerL30Ala / LysL31Ala;         L2, SerL50Ala / SerL52Ala / ThrL53Ala;         L3, AsnL92Ala / GluL93Ala / TyrL94Ala.         ehu4D5 is a humanized anti-p185^HER2Antibody, huMHM 24 antibody (Carter et al., Proc. Natl. Acad. Sci. USA8 9: 4285 (1992)). .   In the heavy chain, CDR-H2 and CDR-H3 make significant contributions to binding You are. The TrpH33Ala residue of CDR-H1 has a great effect, Conformational change because TrpH33 should be partially buried It seems to be due to. The single residue most important for binding is CDR-H Asp-H54 in 2; replacing this residue with alanine gives 1 for binding. A 47-fold reduction occurs (Table IV). Other residues involved in binding in CDR-H2 include , GluH56, GlnH61, and LysH64 (Table IV). CD In R-H3, TyrH97Ala reduces binding by 11-fold and TyrH97Ala H100cAla reduced binding by 8-fold. Multiple CDRs such as CDR-L3 Simultaneously changing the DR-H3 residue to alanine, non-additive Large changes occurred (mutant H3, TyrH97Ala and TyrH99Al See contrast to a; Table IV). Furthermore, FR- contained in the humanized product The LysH73 residue at 3 was also converted when converted to alanine or arginine. Combination Showed a 5-fold reduction for (Table IV).   (C) Re-modification of HuIgG1 to bind to rhesus monkey CD11a   Mouse MHM24 and HuIgG1 both bind to rhesus monkey CD11a About 1000-fold reduction: HuIgG1 was associated with rhesus monkey CD11a. Has an EC50 of 45.6 ± 40.4 nM (N = 16), whereas The EC50 for human CD11a is 0.042 ± 0.072 nM. M Because the primate model is important in the biology, toxicity and efficacy of HM24, Improving the binding of HuIgG1 to rhesus monkey CD11a would be beneficial. It seems to be that. Initially important for rhesus monkeys, but important for humans The human variable in the hypervariable region residues of the MAb so that CD11a and what is important for rhesus monkey binding to CD11a have been determined . Therefore, the alanine-scan mutant was also used for rhesus monkeys on peripheral blood lymphocytes. The assay was performed against CD11a. The most important finding is that multiple alanine- The H2 mutant, one of the mutant strains, is 18 times better than HuIgG1 It bound to CD11a (Table IV). However, included in the H2 mutant Individual mutations at the three residues shown show minimal improvement in binding : HisH52Ala, 0.7 times improvement, SerH53Ala, 0.7 times improvement, And SerH55Ala, 1.3-fold worse (Table IV). In these three residues A series of double mutations is most likely to be the HisH52Ala-SerH53A1a combination. Demonstrated good, but provided a 77-fold improvement over HuIgG1 (See variants H2A1, H2A2 and H2A3, Table IV). Furthermore, H AspH54 in uIgG1 is the most important binding for human CD11a Residues, the AspH54Ala and GluH56Ala mutants also It showed a 3-fold improvement over IgG (Table IV).   Improves Rhesus Monkey Binding to CD11a but Reduces Human CD11a Binding To find a single substitution at site H54 that would not Was replaced with various amino acids. For all replacements, at least one order of magnitude The binding was reduced by AspH54 Asn substitution, whereas the binding of rhesus monkeys was Case was improved by a factor of 10 (Table V).                                   Table V                     Amino acid substitutions at AspH54        a EC50 of HuIgG1 against human CD11a = 0.042 nM (SD = 0.072; N = 15); HuIg against rhesus monkey CD11a EC50 for G1 = 45.6 nM (SD = 40.4; N = 16).         b values are the average of two assays.         c values are the average of a single assay.         For mutations at sites H52-H53, a non-additive effect was seen, These were combined with various changes at sites H54 and H56 (Table VI). For all mutants, H52 and H53 were alanine. One Silly In addition, site H54 is Asn and site H56 is Glu (original). , Ala, Asn, or Gln. All of these mutants have H2A1 mutations. It did not show improved binding to rhesus monkey CD11a harboring variants (Table V I). In another series, site H54 is Ala and H56 is Glu ( Original), Ala, Ser, or Asn, again worse than H2A1 Was. In the third series, site 54 is Ser and site H56 is Gl u (original), Ala, Ser, or Asn. Of these mutants Are improved against rhesus monkey CD11a compared to H2A1 mutant (H2C11 and H2C12, Table VI). These two mutants The EC50 of rhesus monkey CD11a was 0.11 ± for H2C11. 0.11 nM (N = 9) and 0.19 ± 0.08 (N = 7). These values are based on the EC5 of HuIgG1 against human CD11a. Rhesus monkey CD1 which is 3 to 5 times weaker than 0 (0.042 nM) 24.0-fold higher than the EC50 of HuIgG1 against 1a (45.6 nM). A 15-fold improvement. H2C12 is hereinafter referred to as RhIgG1. Saturated bond Apparent K from the experiment_dValues indicate that mouse MHM23 bound to rhesus monkey CD18 Shows that RhIgG1 binds to rhesus monkey CD11a (Table III).                                  Table VI         Binding of CDR-H2 to human and rhesus monkey CD11a        a EC50 of HuIgG1 against human CD11a = 0.042 nM (SD = 0.072, N = 15); HuIg against rhesus monkey CD11a EC1 of G1 = 45.6 nM (SD = 40.4; N = 16); Rhesus monkey All values for CD11a are two independent linkages, unless otherwise noted Mean of the assay.   For the interaction of HuIgG1-human CD11a, AspH54 is the most severe. Key residues (Table IV); replacing this residue with another amino acid significantly bound , But among them, Glu, Asn, and Gln are the best alternatives. Even the decline was low. However, the interaction of HuIgG1-rhesus monkey CD11a In the case of action, AspH54 shows that binding of this residue to Ala or Asn Harmful as improved (Table V). Binding of human and rhesus monkey CD11a Cloned from a rhesus monkey PBL library to understand the differences between It has become. FIG. 2 shows that the rhesus monkey CD11aI-region corresponds to the human CD11aI-region. Differ only in the four regions: 133, 189, 197, 308 Are shown. Previously, a human CD11a epitope on MHM24 was identified at residue 197 -203 (Champe et al., J. Biol. Chem. 2 70: 1388-1394 (1995)), including humans in rhesus monkeys. Changes from Lys197 to rhesus monkey Glu197 are included.   (D) Keratinocyte cell attachment assay   Mouse MHM24, chimeric IgG1 and HuIgG1 were transferred to Jurkat cells ( LFA-1 expressing human T-cells) on a normal human expressing ICAM-1. The ability to block adhesion to skin keratinocytes was compared. All three antibodies , With similar IC50 (FIG. 3) (Table VII).                                  Table VII                   Inhibition of cell adhesion by MHM24 mutant         a HuK = normal human epithelial keratinocytes         b RhLy = Rhesus monkey lymphocyte         c HuICAM = recombinant human ICAM-1         dRh / HuCD11a = human CD11 with rhesus monkey I-region a transfected into human 293 cells   Both mouse and humanized MHM24 were used to control rhesus and cynomolgus monkeys. It did not prevent the adhering spheres from attaching to human keratinocytes. Human keratin rhino In inhibiting rhesus monkey lymphocyte adhesion to Mouse anti-human CD18 antibody MHM23 (Hildreth et al., Eur. Immunol. 13: 202-208 (1983): Hildreth et al., J. . Immunol. 134: 3272-3280 (1985)) RhIgG1 is 74 times less effective than MHM23 (FIG. 4A, Table VII). I However, recombinant human ICAM-1 was pre-purified (instead of human keratinocytes). When coated on a plate, RhIgG1 is only 4 times less effective than MHM23. (FIG. 4B, Table VII). Human CD11a (I-region mutated to rhesus monkey) V al133Ile, Arg189Gln, Lys197G1u, Val308A l) transfecting chimeric CD11a with human embryonic kidney 293 cells did. Human keratinosa for these Rh / HuCD11a-293 cells RhIgG1 is again more effective than MHM23 in blocking adhesion to 4 times lower (FIG. 4C, Table VII).   A control isotype antibody against RhIgG1 (humanized anti-p185^HER2Anti Body; Carter et al., Proc. Natl. Acad. Sci. USA 89: 4 285 (1992)) and MHM23 (mouse MAb354, mouse IgG1 Anti-hamster tpA) is a rhesus monkey lymphocyte against human keratinocytes Binding was also due to recombinant ICAM-1 to human keratinocytes (FIGS. 4A, 4B), and It also did not block Rh / HuCD11a (FIG. 4C) binding. This is a Rhesus monkey lymphocytes: ratio with mouse MHM23 in human keratinocyte assay The reduced performance of RIgG1 when compared to (MHM23 mouse Fc Interaction of HuIgG1 with rhesus monkey lymphocytes (when performed) Suggest that this is not due to the use of rhesus monkey CD11a. It may reduce the concentration of RhIgG1 capable of binding. Recombinant human IC The data for AM-1 indicate that RhIgG1 binds to rhesus monkey lymphocytes, 4 shows that adhesion is inhibited almost similarly to MHM23 (FIG. 4B, Table VII). The data for Rh / HuCD11a-293 (FIG. 4C, Table VII) RhIgG1 does not bind to targets on human keratinocytes (HuIgG1 1) when compared to Rh capable of binding to rhesus monkey CD11a. May reduce the concentration of IgG1. Furthermore, against rhesus monkey IgG1 RhIgG1 K_d(App) is 1 mg / ml human IgG1 (Rhesus monkey) In the case of donor 3), it was similar to the case without (rhesus monkey donor 1) (Table II). I). This indicates that the binding of RhIgG1 is specific for rhesus monkey CD11a. Are shown.   (E) Mixed lymphocyte response assay   In the MLR, HuIgG1 has an IC50 that is twice as weak as mouse MHM24. The values are shown (Table VIII, FIG. 5).                                Table VIII                      Mixed lymphocyte response assay results         a murMHM24, HuIgG1 and mAb 25.3 are human ML Tested on R: RhIgG1 and MHM23 were tested on rhesus monkey MLR.   Mouse and humanized MAbs are 10- to 20-fold greater than MAb 25.3 Worked, but MAb 25.3 has been previously tested in vivo ( Fisher et al., Blood 77: 249-256 (1991); Stoppa. Et al., Transplant Intl. 4: 3-7 (1991); Hourma. nt et al., Transplantation, 58: 377-380 (1994). ). Rhesus monkey-binding mutant RhIgG1 is slightly less than mouse MHM23 A good IC50 is shown (Table VIII). Different responders: stimulator blood donor Was used in an independent assay, but the results were not significantly different. A RhIgG1 K against rhesus monkey CD11a_dIs H to human CD11a 26-fold lower than the Kd of uIgG1 (Table III), derived from the MLR assay (Table VIII).

[Procedure amendment] [Submission date] May 28, 1999 (1999.5.28) [Content of amendment] Claims 1. A humanized anti-CD11a antibody that specifically binds to the human CD11aI-domain. 2. The humanized anti-CD11a antibody of claim 1, which binds to the epitope MHM24 on CD11a. 3. 3. The humanized anti-CD11a antibody of claim 1 or 2 having human Kappa I consensus light chain framework residues 66L . 4. 4. The humanized anti-CD11a antibody of any one of claims 1-3 , having all human kappa I consensus light chain framework residues . 5. Human V _H subgroup III consensus heavy chain framework residues 93H to have a billing humanized anti CD11a antibody according to any one of claim 1 to 4. 6. The _VH subgroup III consensus contains a variable domain with a non- human CDR incorporated within a human antibody variable domain and further comprises amino acid substitutions at a site selected from the group consisting of 27H, 28H, 30H, 49H , 71H and 73H. Bei obtain claims 1 to 5 any one of claims humanized anti CD11a antibody of. 7 . Claims having a heavy chain variable region comprising the amino acid sequence of CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and / or CDR3 (SEQ ID NO: 12) of humanized antibody MHM24 (F (ab) -8) Item 3. A humanized anti-CD11a antibody according to claim 1 or 2. 8. A humanized anti-CD11a antibody according to claim 1 or 2, which comprises the amino acid sequence of SEQ ID NO: 5. 9. A humanized MHM24 (F (ab) -8) The humanized anti-CD11a antibody according to claim 1 or 2, which has a light chain variable region including an amino acid sequence of CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and / or CDR3 (SEQ ID NO: 15). . 10 SEQ ID NO: claim 1 or 2, wherein the humanized anti-CD 11a antibody comprising amino acid sequence 1 1 SEQ ID NO:.. and a light chain variable region comprising the amino acid sequence of the 5, SEQ ID NO: 2 amino acids Light chain variable region containing sequence .... Claim 1 or 2, wherein the humanized anti-CD1 1a antibody having 1 2 full length antibodies or humanized anti-CD11a antibody according to any one of the antibody fragment is a claims 1 to 11 13 following characteristics: (A) binds to human CD11a with a K _d value up to about 1 × 10 ⁻⁸ M; (b) IC50 (nM up to about 1 nM to prevent adhesion of Jurkat cells to normal human epidermal keratinocyte-expressed ICAM-1 ) has a value, or (c) mixing have a IC50 (nM) values of up to about 1nM in lymphocyte response assay as claimed any one of claims 1 to 12 having at least one humanized anti-CD 11a antibody. 1 4. detectable with a humanized anti-CD11a antibody of any one of claims 1 to 13 bound to a label labeled antibody. 1 5. immobilized on a solid phase Claims 1 to 13 immobilized antibodies with humanized anti-C D11a antibody according to any one of. 1 6. Humanization of any one of cytotoxic agents from claim 1 engaged binding to 13 conjugate comprising an anti-CD11a antibody. 1 7. exposing a sample expected to contain the humanized anti CD11a antibody CD 11a proteins according to any one of claims 1 to 13, and the antibody to said sample and methods. 1 8. of any one of claims 1 to 13 human estuary CD11a antibodies for determining the presence of CD11a protein comprising measuring the binding, the antibody to detect the C D11a protein Kit with instructions for use. 19 . An isolated nucleic acid encoding the humanized anti-CD11a antibody according to any one of claims 1 to 13 . 2 0. A vector comprising the nucleic acid according to claim 19 . 2 1. Host cell comprising the vector of claim 2 0, wherein. 2 2. Culturing a host cell according to claim 2 1, wherein such nucleic acid is expressed; and a method for manufacturing human anti CD11a antibody comprising recovering the humanized anti-CD11a antibody from the host cell culture. 23 . The order to prepare a medicament for the treatment of patients with LFA-mediated disease in its necessary use of humanized anti-CD11a antibody according to any one of the preceding top 1 13.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 G01N 33/53 D G01N 33/53 C12P 21/08 // C12P 21/08 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ) , BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (1)

[Claims] 1. A humanized anti-CD11a antibody that specifically binds to the human CD11aI-domain. 2. The humanized anti-CD11a antibody of claim 1, which binds to the epitope MHM24 on CD11a. 3. A humanized anti-CD11a antibody that binds to human CD11a with a K _d value of up to about 1 × 10 ⁻⁸ M. 4. A humanized anti-CD11a antibody having an IC50 (nM) value of up to about 1 nM to prevent adhesion of Jurkat cells to normal human keratinocyte expressing ICAM-1. 5. A humanized anti-CD11a antibody having an IC50 (nM) value of up to about 1 nM in a mixed lymphocyte response assay. 6. 2. A heavy chain variable region comprising an amino acid sequence of CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the humanized antibody MHM24 (F (ab) -8). 7. The humanized anti-CD11a antibody according to claim 7. 7. The humanized anti-CD11a antibody according to claim 6, comprising the amino acid sequence of SEQ ID NO: 5. 8. The CDR1 of humanized MHM24 (F (ab) -8 (SEQ ID NO: 13)) 2. A humanized anti-CD11a antibody according to claim 1, which has a light chain variable region comprising the amino acid sequences of CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) 9. Includes the amino acid sequence of SEQ ID NO: 2 9. The humanized anti-CD11a antibody according to claim 8. 10. The humanized antibody according to claim 1, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. Anti-CD11a antibody 11. The humanized anti-CD11a antibody according to claim 1, which is a full-length antibody 12. The humanized anti-CD11a antibody, according to claim 11, which is human IgG 13. The antibody according to claim 1, which is an antibody fragment. 14. The antibody fragment of claim 13, which is F (ab ') _2. 15. The antibody of claim 1, labeled with a detectable label 16. The antibody of claim 1, immobilized on a solid phase. 18. The antibody of claim 1, conjugated to a cytotoxic agent 18. Exposure to a sample expected to contain the CD11a protein to the antibody of claim 1, and measuring the binding of the antibody to the sample. 19. A method for measuring the presence of the CD11a protein 19. A kit comprising the antibody according to claim 1 and instructions for using the antibody for detecting the CD11a protein. 20. An isolated nucleic acid encoding the antibody of claim 1. 21. A vector containing the nucleic acid of claim 20. 22. A host cell containing the vector of claim 21 23. Claiming that the nucleic acid is expressed Item 23. A method for producing a humanized anti-CD11a antibody, comprising culturing the host cell according to item 22. 24. The method according to claim 23, further comprising recovering the humanized anti-CD11a antibody from the host cell culture. 25. The method of claim 24, wherein the anti-CD11a antibody is recovered from the host cell medium.